Endocytosis agent

Abstract

Disclosed herein are compounds comprising an agent or probe linked to a chemical arm via a cleavable or non-cleavable chemical bond or linker unit, wherein the compounds have a binding affinity for endocytosis-mediated membrane components. Methods of using and making the compounds are also disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 420,326, filed on October 28, 2022, the contents of which are incorporated by reference in their entirety. Background Art

[0003] According to traditional guidelines, such as the "Rule of 5" (Ro5) and its subsequent extensions (extended Ro5; eRo5 and beyond Ro5; bRo5), most therapeutic agents have poor drug-likeness due to unfavorable stability, water solubility, and / or membrane permeability, directly leading to high attrition rates in drug development. Among these factors, poor permeability has been considered the most important issue, as drugs must cross numerous membrane barriers to reach the site of drug action after administration. Based on passive permeation, stability, water solubility, and membrane permeability are highly correlated and sensitive to structural changes, and structural modifications used to adjust one property will inevitably affect one or more other properties. To increase drug passive permeability, a common approach is to enhance the lipophilicity of the compound through specific structural optimization, making it easily soluble in membrane phospholipids and rapidly diffusing across cell membranes, but at the cost of reduced solubility (due to reduced hydrophilicity), stability, and / or increased toxicity. For most therapeutic agents, favorable stability and water solubility can be achieved through structural modification and salt formation techniques. However, how to improve the membrane permeability of therapeutic agents without sacrificing solubility and stability has become the ultimate issue that needs to be addressed in traditional drug discovery and development activities. Evidence shows that the passive permeability of drugs decreases dramatically as the size of the molecule increases. Unfortunately, the biological activity of eRo5 and bRo5 molecules (such as chimeric molecules) has been attributed to passive permeability, and the optimization of eRo5 and bRo5 molecules has been based on passive diffusion, although according to passive diffusion theory, it is challenging or even impossible to modify macromolecules (such as PROTAC molecules) to improve membrane passive permeability while balancing good metabolic stability and solubility. Therefore, there is a need in the art for a general method that can be used to accelerate the development of therapeutic agents and broaden the therapeutic applications of pharmaceutical agents by enhancing permeability without sacrificing solubility and stability. Summary of the Invention

[0004] Disclosed herein are compounds comprising an agent or probe connected to a chemical arm via a cleavable or non-cleavable chemical bond or linker unit, wherein the compound has a binding affinity for a membrane component that mediates endocytosis. The compound may have a binding affinity KD of less than 20.0 mM for the membrane component that mediates endocytosis. In some embodiments, the compound is represented by formula (I): wherein m, n, and p represent integers from 0 to 100. Exemplary compounds may have m, n, and p equal to 1. The compound may comprise an agent that is a degrader, stabilizer, inhibitor, modulator, or activator. In some embodiments, the agent is a protein binder. In some embodiments, the compound comprises a probe that is a diagnostic agent. The chemical arm may be an atom, an agent, a probe, or a portion of an agent or a binder or a probe. In some cases, the binding affinity K of the chemical arm for the membrane component that mediates endocytosis is D Less than 20.0 mM. Suitably, the membrane component mediating endocytosis is a cell membrane lipid, carbohydrate or protein. The membrane component mediating endocytosis can be a glycolipid, glycoprotein, phospholipid, ceramide and cholesterol. In some embodiments, the membrane component mediating endocytosis is a glycolipid or glycoprotein comprising 2-100 linear or branched monosaccharide units. The membrane component mediating endocytosis can be an integral membrane protein, a peripheral membrane protein, a lipid-anchored protein, a globular protein or a glycoprotein.

[0005] Also disclosed are nanostructures comprising a liquid or cytoplasm enclosed by a lipid bilayer, a membrane component that mediates endocytosis, and any compound described herein. In some cases, the nanostructure has a binding affinity, K, for the membrane component that mediates endocytosis. D Less than 20.0 mM. Exemplary nanostructures include extracellular vesicles, microvesicles, endocytic agent-vesicle complexes, or exocytic vesicles.

[0006] Also provided are methods for preparing nanostructures. The methods may include contacting any compound disclosed herein with cells or vesicles comprising the membrane component that mediates endocytosis. The nanostructures may be prepared in vivo, in vitro, or ex vivo.

[0007] Also provided are methods for internalizing any compound disclosed herein within a cell. The methods may include contacting any compound disclosed herein with a cell comprising a membrane component that mediates endocytosis. In some embodiments, a nanostructure comprising the compound is contacted with the cell.

[0008] A method for separating the compound from the nanostructure is also provided. The method may include lysing the nanostructure and separating the compound from the lysate.

[0009] Also provided are methods for determining the qualitative or quantitative presence of a compound or nanostructure in a cell, body, liquid, or culture medium. The methods may include centrifuging a sample containing the cell, body, liquid, or culture medium and detecting the compound or nanostructure.

[0010] Also provided is a method for isolating the compound or nanostructure from a cell, body, liquid, or medium. The method may include centrifuging a sample containing the cell, body, liquid, or medium.

[0011] Also provided is a method for treating a subject. The method may comprise administering a compound or nanostructure to a subject in need thereof.

[0012] Also provided is a method for identifying an endocytosis agent. The method may include contacting a compound with a first cell and a second cell, wherein the presence of a membrane component mediating endocytosis in the second cell is modulated relative to the first cell, and comparing the activity of the compound in contact with the first cell with the activity of the compound in contact with the second cell to determine that the compound is the endocytosis agent.

[0013] Also provided is a method for identifying a membrane component that mediates endocytosis. The method can include contacting a cell with the compound or nanostructure, wherein the compound comprises a detectable label and wherein the membrane component that mediates endocytosis in the cell can be identified by determining an interaction between the membrane component that mediates endocytosis and the detectable label.

[0014] Also provided is a method for identifying a membrane component that mediates endocytosis. The method may include comparing the sensitivity of a first cell and a second cell to treatment with a compound or nanostructure, and comparing the genomic expression or protein abundance of the membrane component in the first cell and the second cell, wherein increased sensitivity of the first cell or the second cell to the compound or nanostructure identifies the membrane component that mediates endocytosis.

[0015] Also provided is a method for selecting a subject for treatment with a compound or nanostructure having binding affinity for a membrane component that mediates endocytosis. The method may include determining the qualitative or quantitative presence of the membrane component that mediates endocytosis in a sample obtained from the intended subject, and administering the compound or nanostructure to the intended subject when the membrane component that mediates endocytosis is present in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying drawings, which are schematic and not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is generally represented by a single numeral. For clarity purposes, not every component is labeled in each figure, nor is every component of each embodiment of the present invention shown where no graphical illustration is needed to allow one of ordinary skill in the art to understand the present invention.

[0017] Figure 1A-1E Schematic illustration of endocytic agents and their utilization.

[0018] Figure 1A Delineating the mechanisms and utility of cellular pickup of endocytic agents and methods for generating endocytic agents or polypharmacological endocytic agents with increased binding affinity and / or valency to membrane components mediating endocytosis to enhance endocytic efficacy and / or efficiency.

[0019] Figure 1B Describe the mechanisms by which endocytic agents are absorbed into the blood following oral, inhalation, or topical administration. Endocytic agents can be encapsulated in intracellular vesicles and released into the bloodstream as free endocytic agents and / or exocytic vesicles via a variety of pathways. Cytoplasmic proteins include, but are not limited to, fatty acid binding proteins (FABPs).

[0020] Figure 1C Describe the mechanism by which endocytic agents are taken up from the bloodstream into brain tissue by crossing the blood-brain barrier. Endocytic agents can be encapsulated in intracellular vesicles and released into the bloodstream as free endocytic agents and / or exocytic vesicles via a variety of pathways.

[0021] Figure 1D Describe the mechanisms by which cells pick up, release, and utilize endocytic and exocytic vesicles to carry out biological functions.

[0022] Figure 1E Plotted are the relative permeabilities of agents with a range of molecular weights via passive diffusion (top curve at low molecular weight) or endocytosis (bottom curve at low molecular weight).

[0023] Figure 2A-2E Endocytotic agents were shown to bind to human CD36 protein.

[0024] Figure 2A Depicts immunoblotting assay demonstrating expression of CD36 protein in the membrane and cytoplasmic compartments of LNCaP PCa cells.

[0025] Figure 2B Depicts a pull-down assay demonstrating that CD36 protein in the LNCaP cell membrane is one of the targets of a polypharmacological endocytic agent as exemplified by Example 1. Mass spectrometry (MS) confirmed the binding of Example 1 to the bromodomain and extraterminal (BET) protein family, CD36, as well as proteins associated with endocytic vesicle formation, tethering, fusion, trafficking, and recycling of membrane components, such as CLH1, UBR4, AP1B1, RAB5A, BIG1, AP2B1, AP3D1, ARP2, and AP3B1.

[0026] Figure 2CThe endocytosis agent shown in Example 1, Example 3, or Example 4 binds to the hCD36 protein. Immunoblotting of CD36 after treatment of purified his-tagged hCD36 protein with a biotin control or an endocytosis agent example.

[0027] Figure 2D Multiple pharmacological endocytic agents, as exemplified by Example 6 or Example 7, were shown to bind to hCD36 protein. Immunoblotting for CD36 following treatment of purified his-tagged hCD36 protein with biotin control or endocytic agent examples.

[0028] Figure 2E The representative endocytosis agent Example 8 is shown as binding to human CD36 protein as determined by ITC assay. The titration consisted of injecting Example 8 solution from a syringe into a solution of human CD36 protein (hCD36) in a sample cell at a rate of 0.5 μl / s at 150 s intervals. K was determined using the sequential binding site model. D value.

[0029] Figure 3A-Figure 3O This indicates that endocytosis is the major route of drug uptake.

[0030] Figure 3A Shown are CD36 protein levels in LNCaP PCa cells transfected with shRNAs harboring Luc control or CD36-targeting sequences.

[0031] Figure 3B Shown are EEA1 protein levels in LNCaP cells transfected with shRNAs with Luc control or EEA1-targeting sequences.

[0032] Figure 3C Shown are Rab5 protein levels in LNCaP cells transfected with shRNAs with Luc control or Rab5-targeting sequences.

[0033] Figure 3D Shown are CD36 protein levels in 22Rv1PCa cells transfected with siRNAs with a scrambled sequence or a CD36-targeting sequence.

[0034] Figure 3E Demonstrating that EEA1 / Rab5 proteins are required for endocytosis and promote transferrin uptake. Representative confocal images of DAPI and transferrin morphology in LNCaP cells treated with stabilized shLuc, shEEA1, or shRab5 for the indicated times. Scale bar: 100 μM.

[0035] Figure 3FColocalization assays are shown demonstrating the dependence of multiple pharmacological endocytic agent uptake in LNCaP cells on CD36-mediated endocytosis, as exemplified by Example 10. Following treatment of LNCaP cells with Example 10 for the indicated durations, fluorescence from the endocytic agent Example 10 and DAPI in the cells was imaged on a fluorescence microscope. Scale bar: 200 μm.

[0036] Figure 3G Colocalization assays are shown demonstrating the dependence of trivalent multipharmacological endocytic agent uptake in LNCaP cells on CD36-mediated endocytosis, as exemplified by Example 12. Following treatment of LNCaP cells with Example 12 for the indicated durations, fluorescence from the endocytic agent Example 12 and DAPI in the cells was imaged on a fluorescence microscope. Scale bar: 100 μM.

[0037] Figure 3H Colocalization assays are shown demonstrating the dependence of trivalent multipharmacological endocytic agent uptake in LNCaP cells on CD36-mediated endocytosis, as exemplified by Example 8. After treatment of LNCaP cells with Example 8 for the indicated durations, fluorescence from the endocytic agent Example 8 and DAPI in the cells was imaged on a fluorescence microscope. Scale bar: 100 μM.

[0038] Figure 3I Colocalization assays were performed to demonstrate the dependence of trivalent multipharmacological endocytic agent uptake in 22Rv1 cells on CD36-mediated endocytosis, as exemplified by Example 8. After 22Rv1 cells were treated with 500 nM of Example 8 for 0.5 h, fluorescence from the endocytic agent Example 8, CD36 protein, EEA1 protein, and DAPI in the cells was imaged on a fluorescence microscope.

[0039] Figure 3J Colocalization assays are shown demonstrating the dependence of LNCaP cells on CD36-mediated endocytosis for the uptake of multiple pharmacological endocytic agents, as exemplified by Example 13. Following treatment of LNCaP cells with Example 13 for the indicated durations, fluorescence from the endocytic agent Example 13 and DAPI in the cells was imaged on a fluorescence microscope. Scale bar: 200 μm.

[0040] Figure 3K Colocalization assays are shown demonstrating the dependence of bivalent multipharmacological endocytic agent uptake in LNCaP cells on CD36-mediated endocytosis, as exemplified by Example 14. Following treatment of LNCaP cells with Example 14 for the indicated durations, fluorescence from the endocytic agent Example 14 and DAPI in the cells was imaged on a fluorescence microscope. Scale bar: 100 μM.

[0041] Figure 3LColocalization assays are shown demonstrating the dependence of LNCaP cells on CD36-mediated endocytosis for the uptake of bivalent multipharmacological endocytic agents, as exemplified by Example 15. Following treatment of LNCaP cells with Example 15 for the indicated durations, fluorescence from the endocytic agent Example 15 and DAPI from the cells was imaged on a fluorescence microscope. Scale bar: 100 μM.

[0042] Figure 3M Colocalization assays demonstrate the dependence of bivalent multipharmacological endocytic agent uptake in 22Rv1 cells on CD36-mediated endocytosis, as exemplified by Example 15. After 22Rv1 cells were treated with 500 nM of Example 15 for 0.5 hours, fluorescence from the endocytic agent Example 15, CD36 protein, EEA1 protein, and DAPI was imaged on a fluorescence microscope. Scale bar: 100 μM.

[0043] Figure 3N Colocalization assays are shown demonstrating the dependence of 22Rv1 cells on CD36-mediated endocytosis for the uptake of bivalent multipharmacological endocytic agents, as exemplified by Example 16. After treatment of 22Rv1 cells with Example 16 for the indicated durations, fluorescence from the endocytic agent Example 16 and DAPI from the cells was imaged on a fluorescence microscope. Scale bar: 100 μM.

[0044] Figure 3O The trivalent multipharmacological endocytic agent Example 8 or Example 12 and the bivalent multipharmacological endocytic agent Example 14 or Example 15 were shown to have little or no observable toxicity on LNCaP or 22Rv1 cell viability (n=3).

[0045] Figures 4A-4F CD36-mediated endocytosis was shown to determine drug uptake and biological function.

[0046] Figure 4A The multipharmacological endocytic agent trivalent-BETD1 (Example 17) was shown to induce BRD4 degradation in LNCaP or 22Rv1 PCa cells. The levels of BRD4 protein in LNCaP or 22Rv1 cells treated with Example 17 with or without MG132 pretreatment were determined by western blotting.

[0047] Figure 4B shCD36 in LNCaP or 22Rv1 PCa cells was shown to reverse BRD4 degradation caused by the multipharmacological endocytosis agent trivalent-BETD1 (Example 17). LNCaP or 22Rv1 cells stably transfected with shLuc or shCD36 were treated with Example 17, and the protein levels of BRD4 and CD36 were determined by Western blot assay.

[0048] Figure 4C The multipharmacological bivalent endocytic agent ARV110 (Example 19) was shown to induce AR degradation in LNCaP or 22Rv1 PCa cells. The protein levels of AR in LNCaP or 22Rv1 cells treated with Example 19 with or without MG132 pretreatment were determined by Western blot assay.

[0049] Figure 4D shCD36 in LNCaP or 22Rv1 PCa cells was shown to reverse AR degradation caused by the multipharmacological bivalent endocytosis agent ARV110 (Example 19). LNCaP or 22Rv1 cells stably transfected with shLuc or shCD36 were treated with Example 19, and the protein levels of AR and CD36 were determined by Western blotting.

[0050] Figure 4E and Figure 4F The anti-tumor effect of the shCD36 antagonist multipharmacological endocytogen ARV110 (Example 19) in the LNCaP xenograft mouse model was demonstrated (n=5). Figure 4E , the top curve represents shCD36 no. 2+ARV110; the second curve from the top represents shCD36 no. 2; the third curve from the top represents shLuc+vehicle; the fourth curve from the top represents shCD36 no. 1+ARV110; the fifth curve from the top represents shCD36 no. 1; and the bottom curve represents shLuc+ARV110.

[0051] Figures 5A-5D Conjugation of agents to the chemical arm was shown to enhance cellular uptake via CD36-mediated endocytosis.

[0052] Figure 5A Conjugating an agent (Example 20) to a dicarboxylic acid chemical arm enhances and accelerates its cellular uptake. HCC1806 cells were treated with 500 nM of Example 20 or Example 21 for the indicated durations. Green fluorescence from the endocytosed agents, divalent-BDP-FL1 (Example 20) or trivalent BDP-FL1-C12Na (Example 21), was imaged on a fluorescence microscope. Scale bar: 100 μM.

[0053] Figure 5BColocalization assays demonstrate that CD36-mediated endocytosis is the primary pathway for uptake of the endocytic agents divalent-BDP-FL1 (Example 20), trivalent-BDP-FL1-C12Na (Example 21), or trivalent-BDP-FL1-C14Na (Example 22) in HCC1806 cells. After treatment of HCC1806 cells with 500 nM of Example 20 or Example 21 for 0.5 hours, fluorescence microscopy was performed to image the endocytic agents Example 20, Example 21, or Example 22, CD36 protein, EEA1 protein, and DAPI from the cells. Examples 21 and 22, which have higher binding affinity and more binding arms (valency) than Example 20, enhance and accelerate cellular uptake. Scale bar: 100 μM.

[0054] Figure 5C Colocalization assays demonstrate that CD36-mediated endocytosis is the primary pathway for uptake of the endocytic agents divalent-BDP-FL1 (Example 20), trivalent-BDP-FL1-C12Na (Example 21), or trivalent-BDP-FL1-C14Na (Example 22) in shCD36 HCC1806 cells. After treatment of shCD36 HCC1806 cells with 500 nM of Example 20 or Example 21 for 0.5 hours, fluorescence from the endocytic agents Example 20, Example 21, or Example 22, CD36 protein, EEA1 protein, and DAPI was imaged using a fluorescence microscope. Scale bar: 100 μM.

[0055] Figure 5D It was shown that bivalent-BDP-FL1 (Example 20), trivalent-BDP-FL1-C12Na (Example 21), or trivalent-BDP-FL1-C14Na (Example 22) had almost no cytotoxicity in HCC1806 cells. Meanwhile, trivalent endocytic agents Examples 28 and 29 exerted more potent cytotoxicity in HCC1806 TNBC cells than bivalent endocytic agent Example 23 (n=2).

[0056] Figures 6A-6D CACO2 cells were shown to take up endocytic agents via endocytosis and release free endocytic agents and exocytic vesicles.

[0057] Figure 6A Depicted are representative methods of the present disclosure for isolating and / or detecting endocytic agents and / or exocytic vesicles in culture medium containing cells following treatment with an endocytic agent.

[0058] Figure 6B Depicted are representative methods of the present disclosure for isolating and / or detecting endocytic agents and / or exocytic vesicles in the blood and / or tissues of animals following treatment of the animals with an endocytic agent via oral administration.

[0059] Figure 6CThe multipharmacological bivalent endocytic agent ARV110 (Example 19) was detected in the supernatant or exosome lysate isolated from culture medium containing CACO2 cells after treatment with Example 19. Compared to the exosome lysate samples, Example 19 was not detected or barely detected in the supernatant samples. The endocytic agent was detected in all samples by LC-mass spectrometry.

[0060] Figure 6D Shown are CD36, CD9, HSP70, and TSG101 protein levels in exocytic vesicles released from CACO2 cells after treatment with a DMSO control or the representative multipharmacological bivalent endocytosis agent ARV110 (Example 19). Immunoblotting of CD36, CD9, HSP70, and TSG101 proteins in exosomes isolated from CACO2 cells after treatment with either a DMSO vehicle control or Example 19. Example 19 treatment enhanced CD36 expression in exocytic vesicles compared to the vehicle control.

[0061] Figure 6E Figure 1 shows the protein levels of CD36, CD9, HSP70, and TSG101 in exosomes isolated from rat plasma after oral administration of the indicated doses of vehicle or a representative multipharmacological bivalent endocytic agent, ARV110 (Example 19). Compared to the control vehicle, Example 19 treatment enhanced the expression of CD36 in exocytic vesicles in vivo. Furthermore, Example 19 was detected at higher abundance in all corresponding exosome lysate samples compared to the supernatant samples from rat plasma after treatment with ARV110 by LC-mass spectrometry.

[0062] Figures 7A-7K Increasing binding affinity (see Table 2) and / or binding valency (see Table 1) to CD36 was shown to result in enhanced agent uptake and therapeutic outcome.

[0063] Figure 7A It was shown that under the same conditions, some trivalent endocytic agents with higher binding affinity and more binding arms (valency) (Examples 24 to 31) induced deeper BRD4 degradation in HCC1806 TNBC or 22Rv1 PCa cells than the bivalent endocytic agent (Example 23). Immunoblotting for BRD4 after treatment of HCC1806 or 22Rv1 cells with DMSO vehicle control or endocytic agents.

[0064] Figure 7BIt was shown that under the same conditions, the trivalent endocytosis agent Example 29 with higher binding affinity and more binding arms (valence) induced deeper BRD4 degradation in HCC1806 TNBC cells than the bivalent endocytosis agent (Example 23). BRD4 protein was examined by immunoblotting, and BRD4 protein levels were quantified by densitometry and normalized to the corresponding density of β-tubulin.

[0065] Figure 7C It is shown that under the same conditions, the trivalent endocytogen Example 29 with higher binding affinity and more binding arms (valency) induces faster BRD4 degradation in HCC1806 TNBC cells than the bivalent endocytogen (Example 23). Immunoblotting for BRD4 after treatment of HCC1806 cells with DMSO vehicle control or endocytogen for the indicated durations.

[0066] Figure 7D It was shown that MG132 pretreatment reversed BRD4 degradation in HCC1806 cells caused by the endocytosis agent Example 23 or Example 29. The protein level of BRD4 in HCC1806 cells treated with the indicated concentrations of the endocytosis agent Example (with or without MG132 pretreatment) was determined by Western blotting.

[0067] Figure 7E It was shown that shCD36 reversed the degradation of BRD4 in HCC1806 cells caused by the endocytosis agent Example 23 or Example 29. HCC1806 cells stably transfected with or without shCD36 were treated with the indicated concentrations of the endocytosis agent Example, and the protein levels of BRD4 and CD36 were determined by Western blotting.

[0068] Figure 7F It was shown that pretreatment with a SYK inhibitor known to block endocytosis reversed BRD4 degradation in HCC1806 cells caused by the endocytosis agent Example 23 or Example 29. The protein level of BRD4 in HCC1806 cells treated with the indicated concentrations of the endocytosis agent Example (with or without entospletinib pretreatment) was determined by Western blot.

[0069] Figure 7G It was shown that under the same conditions, some trivalent endocytic agents with higher binding affinity and more binding arms (valency) (Examples 32 to 37) induced deeper BRD4 degradation in HCC1806 TNBC or 22Rv1 PCa cells than the bivalent endocytic agent (Example 23). Immunoblotting for BRD4 after treatment of HCC1806 or 22Rv1 cells with DMSO vehicle control or endocytic agents.

[0070] Figure 7HIt was shown that under the same conditions, the trivalent endocytosis agent Example 35, which has higher binding affinity and more binding arms (valence), induced deeper BRD4 degradation in 22Rv1 PCa cells than the bivalent endocytosis agent (Example 23). BRD4 protein was examined by immunoblotting, and BRD4 protein levels were quantified by densitometry and normalized to the corresponding density of β-tubulin.

[0071] Figure 7I The trivalent endocytogen Example 35, which has higher binding affinity and more binding arms (valency), was shown to induce faster BRD4 degradation in 22Rv1 PCa cells than the bivalent endocytogen (Example 23). Immunoblotting for BRD4 following treatment of 22Rv1 cells with DMSO vehicle control or endocytogen for the indicated durations.

[0072] Figure 7J MG132 pretreatment was shown to reverse BRD4 degradation in 22Rv1 PCa cells caused by endocytosis agent Example 23, Example 35, or Example 36. The protein level of BRD4 in 22Rv1 cells treated with the indicated concentrations of endocytosis agent Example (with or without MG132 pretreatment) was determined by Western blot assay.

[0073] Figure 7K It was shown that shCD36 reversed BRD4 degradation in 22Rv1 PCa cells caused by endocytosis agent Example 23 or Example 35. 22Rv1 cells stably transfected with or without shCD36 were treated with the indicated concentrations of endocytosis agent Example, and then BRD4 protein levels were determined by Western blotting.

[0074] Figures 8A-8H The introduction of chemical arms via cleavable bonds or moieties was shown to enhance agent uptake and therapeutic outcomes.

[0075] Figure 8A It is shown that under the same conditions, some endocytosis agents (embodiments 44 to 51) conjugated to chemical arms via cleavable bonds induce deeper AR protein degradation in MDA-MB-453TNBC cells than endocytosis agent ARV110 (embodiment 19). Unlike the data in MDA-MB-453 cells, embodiments 44 to 51 cannot induce AR degradation in C4-2 PCa cells, indicating that there are isoforms and / or conformational diversity of membrane proteins in different cancer cell lines. Immunoblotting of AR after treating MDA-MB-453 or C4-2 cells with DMSO vehicle control, enzalutamide or embodiment 19 or embodiments 44 to 51.

[0076] Figure 8BIt is shown that under the same conditions, some endocytic agents conjugated to different chemical arms via cleavable bonds (Examples 46-4, 46, 52 or 53) induced deeper AR protein degradation in MDA-MB-453TNBC cells than the endocytic agent ARV110 (Example 19). Immunoblotting of AR after treatment of MDA-MB-453 cells with DMSO vehicle control, enzalutamide or endocytic agent examples.

[0077] Figure 8C It is shown that under the same conditions, the endocytosis agent conjugated to the chemical arm via a cleavable bond (Example 46) induced deeper AR degradation in MDA-MB-453TNBC cells than the endocytosis agent ARV110 (Example 19). AR protein was examined by immunoblotting and AR protein levels were quantified by densitometry and normalized to the corresponding density of GAPDH protein.

[0078] Figure 8D It is shown that under the same conditions, the endocytosis agent conjugated to the chemical arm via a cleavable bond (Example 46) induces faster AR degradation in MDA-MB-453TNBC cells than the endocytosis agent ARV110 (Example 19). Immunoblot of AR after treatment of MDA-MB-453 cells with DMSO vehicle control, Example 19 or Example 46 for the indicated durations.

[0079] Figure 8E ShCD36 was shown to partially reverse AR degradation caused by the endocytosis agent Example 19 or Example 46 in MDA-MB-453 TNBC cells, indicating that CD36 isoforms or other membrane components that mediate endocytosis are involved in endocytosis agent pickup. MDA-MB-453 cells transfected with or without shCD36 were treated with the indicated concentrations of the endocytosis agent Example, and then the protein levels of AR and CD36 were determined by Western blotting.

[0080] Figure 8F MG132 pretreatment was shown to reverse AR degradation in MDA-MB-453 TNBC cells caused by endocytosis agent Example 19 or Example 46. The protein level of AR in MDA-MB-453 cells treated with the indicated concentrations of endocytosis agent Example with or without MG132 pretreatment was determined by Western blot assay.

[0081] Figure 8G It was shown that SYK inhibitor pretreatment reversed AR degradation in MDA-MB-453 TNBC cells as in Example 19 or Example 46. The protein levels of AR in MDA-MB-453 cells treated with the indicated concentrations of the endocytic agent Example with or without entolinib pretreatment were determined by Western blot assay.

[0082] Figure 8HIt is shown that under the same conditions, some endocytic agents conjugated to chemical arms via cleavable bonds (Examples 54 to 61) induce deeper BRD4 protein degradation in HCC1806 TNBC or 22Rv1 PCa cells than the bivalent endocytic agent Example 23. Examples 58 and 59 (see Table 2) with additional binding arms but without enhanced binding affinity induce deeper BRD4 degradation. Some endocytic agents show different potencies in triggering BRD4 degradation in HCC1806 and 22Rv1 cells, indicating the presence of isoforms and / or conformational diversity of membrane proteins in different cancer cell lines. Immunoblotting of BRD4 after treatment of 22Rv1 cells with DMSO vehicle control, Example 23, or Examples 54 to 61.

[0083] Figures 9A-9G The introduction of chemical arms via relatively stable bonds or moieties was shown to enhance agent uptake and therapeutic outcomes.

[0084] Figure 9A Examples 63 to 70 show that some of the multipharmacological trivalent endocytic agents induce deeper BRD4 protein degradation in HCC1806 TNBC or 22Rv1 PCa cells under the same conditions than the multipharmacological bivalent endocytic agent Example 62. Some endocytic agents exhibit different potencies in triggering BRD4 degradation in HCC1806 and 22Rv1 cells, indicating the presence of isoform and / or conformational diversity of membrane proteins in different cancer cell lines. Immunoblotting for BRD4 following treatment of HCC1806 or 22Rv1 cells with DMSO vehicle control or Examples 62 to 70.

[0085] Figure 9B It is shown that under the same conditions, some multipharmacological trivalent endocytic agents Examples 71 to 77 with higher binding affinity and more binding arms (valency) induce deeper BRD4 protein degradation in HCC1806 TNBC, 22Rv1 or LNCaP PCa cells than the multipharmacological bivalent endocytic agent Example 62. Immunoblotting for BRD4 after treatment of HCC1806, 22Rv1 or LNCaP cells with DMSO vehicle control, Example 62 or Examples 71 to 77.

[0086] Figure 9C It is shown that under the same conditions, some multipharmacological trivalent endocytic agents Examples 78 to 85 induce deeper BRD4 protein degradation in 22Rv1 PCa cells than the multipharmacological bivalent endocytic agent Example 62. Immunoblotting for BRD4 after treatment of 22Rv1 cells with DMSO vehicle control, Example 62, or Examples 78 to 85.

[0087] Figure 9DIt is shown that under the same conditions, the multipharmacological trivalent endocytic agent Example 71 with higher binding affinity and more binding arms (valence) induces deeper BRD4 degradation in 22Rv1 cells than the multipharmacological bivalent endocytic agent Example 62. BRD4 protein was examined by immunoblotting, and BRD4 protein levels were quantified by densitometry and normalized to the corresponding density of β-tubulin.

[0088] Figure 9E The multipharmacological trivalent endocytic agent Example 71 was shown to induce faster BRD4 degradation in 22Rv1 cells than the multipharmacological bivalent endocytic agent Example 62. Immunoblotting for BRD4 following treatment of 22Rv1 cells with DMSO vehicle control, Example 62, or Example 71 at the indicated concentrations.

[0089] Figure 9F MG132 pretreatment was shown to reverse BRD4 degradation in 22Rv1 cells caused by the multipharmacological endocytic agent Example 62, Example 71, or Example 79. The protein level of BRD4 in 22Rv1 cells treated with the indicated concentrations of the multipharmacological endocytic agent Example with or without MG132 pretreatment was determined by Western blot assay.

[0090] Figure 9G shCD36 in 22Rv1 cells was shown to reverse BRD4 degradation in 22Rv1 cells caused by the multipharmacological endocytic agent Example 62 or Example 79. 22Rv1 cells stably transfected with or without shCD36 were treated with the indicated concentrations of the multipharmacological endocytic agent Example, and the protein levels of BRD4 and CD36 were determined by Western blotting.

[0091] Figures 10A-10D The introduction of a chemical arm was shown to enhance agent uptake and expand the agent's therapeutic efficacy and safety window.

[0092] Figure 10A The polypharmacological trivalent endocytic agent Example 28 or 29 was shown to exhibit greater anti-tumor efficacy in the breast cancer HCC1806 xenograft mouse model than the polypharmacological bivalent endocytic agent Example 23. In the graph, the top curve represents vehicle; the second curve from the top represents Example 23; the third curve from the top represents Example 28; and the bottom curve represents Example 29.

[0093] Figure 10B The polypharmacological trivalent endocytic agents Example 28 or 29 were shown to have no observable toxicity in the HCC1806 xenograft mouse model. The top curve represents vehicle; the second curve from the top represents Example 23; the third curve from the top represents Example 28; and the bottom curve represents Example 29.

[0094] Figure 10C The polypharmacological trivalent endocytic agent Example 28 or 29 is shown to be more effective than the polypharmacological bivalent endocytic agent Example 23 in reducing BRD4 protein levels in breast cancer HCC1806 xenografts. Immunohistochemistry (brown to black) of BRD4 in representative tumors of mice treated with vehicle, Example 23, Example 28, or Example 29. Scale bar: 20 μm.

[0095] Figure 10D The graph shows that the multipharmacological trivalent endocytic agent Example 36 exhibits greater anti-tumor efficacy in the 22Rv1 prostate cancer xenograft mouse model than the multipharmacological bivalent endocytic agent Example 23. Simultaneously, the multipharmacological trivalent endocytic agent Example 71 or 79 exhibits greater anti-tumor efficacy in the 22Rv1 prostate cancer xenograft mouse model than the multipharmacological bivalent endocytic agent Example 62. In the graph, the top curve represents vehicle; the second curve from the top represents Example 23; the third curve from the top represents Example 62; the fourth curve from the top represents Example 36; the fifth curve from the top represents Example 79; and the bottom curve represents Example 71.

[0096] Figure 10E The multipharmacological trivalent endocytic agents Examples 23, 36, 62, or 71 were shown to have no observable toxicity in the 22Rv1 xenograft mouse model.

[0097] Figure 11 The endocytosis agent conjugated with the chemical arm (Example 29) showed higher solubility in water than the endocytosis agent Example 23. After standing for 24 hours, no precipitation was observed in the aqueous solution of Example 29, and LC-mass spectrometry analysis confirmed the stability of Example 29 in aqueous solution. DETAILED DESCRIPTION

[0098] Disclosed herein are compounds comprising endocytic agents or probes linked to and / or fused to additional membrane-binding moieties. The additional membrane-binding moieties are selected to enhance the efficacy and / or efficiency of endocytosis of membrane components via mediated endocytosis. Linking or fusion of the additional membrane-binding moiety to the endocytic agent or probe allows for greater transmembrane uptake of the endocytic agent than the original agent or probe.

[0099] Also disclosed herein are methods for screening and identifying endocytic agents, including known chemicals with unknown cell-penetrating endocytic mechanisms.

[0100] One benefit of this technology is that it provides a mechanistic basis and method for an alternative and simplified drug discovery and development process based on chemical endocytosis compared to classical drug discovery and development based on passive diffusion. Endocytosis is a biological process by which all cells absorb large-sized and large-volume foreign substances (such as nutrients, viruses, and bacteria) by engulfing them with their cell membrane. The process begins with the recruitment of substances (such as proteins, fatty acids, and exosome particles) that bind to membrane components that mediate endocytosis (such as membrane receptors including transporters). Endocytic pits or invaginations of the plasma membrane are formed by binding to membrane components that mediate endocytosis. Membrane internalization is completed by a rupture process to release early endosomes within the cell. After internalization, the material and endosomal contents can be released within the cell, and the membrane components that mediate endocytosis can be recycled to the cell surface for another round of delivery. In addition, the material and membrane components that mediate endocytosis can be released by the cell to the extracellular compartment in the form of extracellular vesicles (EVs) (such as exosomes) through the process of exocytosis. Compared to passive diffusion driven by a high-to-low concentration gradient until a plateau is reached, the higher efficacy and efficiency of the endocytic process has been demonstrated by the fact that more than 95% of some nutrients in our daily diet are transported by endocytosis and enter the blood circulation (Deanna M. Minich et al., Journal of Lipid Research, 38, 1709-1721, 1997). For example, CD36, a multifunctional scavenger receptor with multiple ligands, is abundantly expressed on the luminal surface of enterocytes in the intestine, promoting CD36-mediated endocytosis as the main pathway for efficient absorption of large amounts of fatty acids in food (Vincenza Cifarelli et al., Comprehensive Physiology, (8) 2, 493-507, 2018; Hélène Poirier et al., European Journal of Biochemistry, 238 (2), 368-373, 1996). The binding of fatty acids to CD36 activates its downstream kinases and triggers endocytosis, effectively promoting the uptake of supplies through membrane barriers (Jian-Wei Hao et al., Nature Communications, 11(1), 4765, 2020; Richard F. Collins et al., Journal of Biological Chemistry, 284(44), 30288-30297, 2009).As revealed by crystallographic studies, a unique feature of CD36 is a large hydrophobic groove (residues 127-279) that spans most of the length of the protein's extracellular domain, allowing for the binding and accommodation of fatty acids such as stearic acid (C18:0) and docosahexaenoic acid (C22:6) (Fu-Lien Hsieh et al., Nature Communications, 7, 12837, 2016; Zineb Tarhda et al., Bioinformatics and Biology Insights, 7, 369-373, 2013). In addition, endocytosis plays many important roles not only in normal cell physiology but also in pathology, such as recycling membrane receptors for dysregulated signaling transduction and providing a steady increase in the influx of exogenous nutrients for the constitutive anabolism of cancer cells. For example, to thrive under stress, cancer cells upregulate the expression of CD36 to increase the production of lipid mass and the generation of oncogenic signaling lipids in cancer cells. Increased expression of CD36 is associated with poor prognosis in various cancer cells such as lung squamous cells, glioblastoma, leukemia, prostate cancer, bladder cancer, and breast cancer (Gloria Pascual et al., Nature, 541, 41-45, 2017; Tian Zhang et al., Blood Cancer Discovery, 1(2), 198-213, 2020; Matthew J. Watt et al., Science Translational Medicine, 11(478), eaau5758, 2019; Aritro Nath et al., Scientific Reports, 5, 14752, 2015; James S. Hale et al., Stem Cell, 32(7), 1746-1758, 2014). However, cellular endocytosis of chemicals for effective and efficient uptake of therapeutic agents by targeting membrane components that mediate endocytosis has not been exploited. Chemical endocytosis approaches accelerate the development of therapeutic agents and expand the range of therapeutic agents that can be effectively used to study, diagnose, prevent, and treat subjects. Broadly speaking, compared to current approaches that seek a balance between conflicting parameters of a drug (stability, water solubility, and membrane permeability), the medicinal chemistry approach based on chemical endocytosis in this technology can simplify and revolutionize the drug discovery and development process simply by improving permeability (via enhancing the efficacy and / or efficiency of endocytosis) and adjusting the metabolic stability of the drug (while maintaining biological activity), because solubility can be improved relatively easily through salt formation or charge formation techniques in the art. In addition, precision health care approaches based on the differential expression of membrane components that mediate endocytosis can be used for patient stratification for personalized selection of drugs and routes of administration of drugs.

[0101] One benefit of this technology is that endocytosis allows for the uptake of agents across membranes. In particular, this technology can be used to allow or increase the uptake of large and / or polar agents that can be effectively used in research, diagnosis, and treatment of subjects. As predicted by the "Rule of 5" (Ro5) (Christopher A. Lipinski et al., Advanced Drug Delivery Reviews, 46, 3-26, 2001), the most influential framework for relating the physicochemical properties of a given compound to its membrane permeability, drugs with smaller size (<500 Da), lower polarity (topological polar surface area (tPSA) ≤ 1000 Da), and higher toxicity (topological polar surface area (tPSA) ≤ 1000 Da) are likely to be endocytosed. ) and higher lipophilicity (but LogP should be ≤ 5) are more likely to cross the cell membrane via passive diffusion. As the molecular size increases (especially MW>700Da), drugs that exhibit higher polarity (higher tPSA value) or lower lipophilicity (lower LogP value) may face a sharp decrease in passive diffusion through the cell membrane ( Matsson et al., Journal of Medicinal Chemistry, 60(5), 1662-1664, 2017; Cameron R. Pye et al., Journal of Medicinal Chemistry, 60(5), 1665-1672, 2017). As disclosed in the present disclosure, endocytic agents having 1) increased binding affinity and / or binding valence to membrane components that mediate endocytosis, 2) allowing or increasing dimerization or clustering of membrane components that mediate endocytosis (Laura Salavessa et al., Proceedings of the National Academy of Sciences of the United States of America, 118(37), e2024893118, 2021; John Maringa Githaka et al., Journal of Cell Science, 129(22), 4175-4189, 2016) or 3) allowing or increasing conformational changes of membrane components that mediate endocytosis can be better taken up into cells via endocytosis.

[0102] As used in the present technology, "clustering" or "cluster" refers to the process by which endocytosis-mediated membrane components assemble into nano- and micron-sized domains to control biological processes, including but not limited to cell adhesion, endocytosis, and immune responses. For example, upon binding to a ligand, clustering of membrane proteins that mediate endocytosis leads to local protein crowding and / or membrane conformational changes, promoting membrane curvature and the initiation of the endocytic process. It will be understood that "clustering" or "clusters" of endocytosis-mediated membrane components include dimerization, oligomerization, and multimerization of membrane components. "Clustering" or "clusters" of membrane components that mediate endocytosis can be homogeneous, heterogeneous, or a combination of homogeneous and heterogeneous mixtures in any ratio. It will also be understood that conformational changes of membrane components that mediate endocytosis can be independent of and / or associated with "clustering" or "clusters" of membrane components that mediate endocytosis.

[0103] After receiving an input signal (such as ligand binding, chemical modification or environmental change), membrane proteins undergo conformational changes, and proteins with different conformations can play different or unique biological functions. For example, after receiving a binding stimulus, membrane proteins can convert conformations and affect the growth, bending and endocytosis of the mesh protein (clathrin) lattice in the cell (KazukiObashi et al., 14,732, Nature Communication, 2023). The conformational changes of membrane proteins caused by ligand binding depend on factors including binding affinity and binding domain (Anna Vangone et al., eLife, 4, e07454, 2015). Increasing the binding valence for picking up additional binding domains can trigger conformational changes and further enhance endocytosis even without enhancing binding affinity. One benefit of the present technology is that it allows or enhances conformational changes in membrane components that mediate endocytosis to enhance endocytic efficacy and / or efficiency through structural modification of endocytic agents, wherein structural modification of endocytic agents includes, but is not limited to, charging the molecule or salt-forming techniques in the art, forming multivalent endocytic agents by linking charged, chargeable, and other hydrophilic chemical arms, or structural modification using medicinal chemistry strategies in the art (including the introduction of reversible or irreversible covalent bonds or moieties). Conformational changes in membrane components that mediate endocytosis for enhancing endocytic efficiency and / or efficacy can be achieved through structural modification of endocytic agents by: 1) increasing the binding affinity and / or binding valence of the endocytic agent to the membrane component that mediates endocytosis, and / or 2) increasing dimerization or clustering of membrane components that mediate endocytosis. In addition, conformational changes in membrane components that mediate endocytosis can be achieved to enhance endocytosis efficiency and / or efficacy through the utilization or structural modification of endocytic agents by regulating: 1) environmental factors of membrane components that mediate endocytosis, such as pH, salinity, oxygen gradients, carbon dioxide gradients, H2O2 gradients, nutrient gradients, and therapeutic compound gradients, 2) interactions with membrane cofactor proteins, and / or 3) post-translational modification states of membrane components that mediate endocytosis and membrane cofactor proteins, such as ubiquitination, phosphorylation, palmitoylation, glycosylation, acetylation, and lipidation.

[0104] Another benefit of this technology is that it allows or increases the permeability of the agent without sacrificing solubility and stability. Based on passive diffusion theory, a common approach to allow or increase the permeability of an agent is to enhance the lipophilicity of the compound through structural optimization based on the specific situation, making it easy to dissolve in membrane phospholipids and diffuse quickly across the cell membrane, but at the expense of reduced solubility (due to reduced hydrophilicity), stability and / or intrinsic biological activity. However, evidence shows that structural optimization of bRo5 agents to allow or increase membrane passive permeability while balancing favorable metabolic stability and solubility is challenging or even impossible ( Matsson et al., Journal of Medicinal Chemistry, 60(5), 1662-1664, 2017; Victoria G. Klein et al., Journal of Medicinal Chemistry, 64(24), 18082-18101, 2021). Compared to the current approach of seeking a balance between the conflicting parameters of therapeutic agents (stability, water solubility and membrane permeability), the present technology simplifies and revolutionizes the drug discovery and development process by simply improving the metabolic stability and endocytosis efficiency of chemical molecules, because water solubility can be achieved relatively easily via techniques such as salt formation. The formation of salts or charges can not only improve solubility, but also increase binding affinity and / or binding valence to endocytosis-mediated membrane components to spontaneously increase cellular uptake. At the same time, increasing water solubility reduces first-pass metabolism and reduces stability risks. The present technology allows for improved solubility and stability characteristics by increasing permeability through enhancing endocytosis efficiency and / or efficacy.

[0105] Another benefit of the present technology is that it allows for increased solubility and / or binding affinity and / or binding valence to membrane components that mediate endocytosis, dimerization or clustering of membrane components that mediate endocytosis, and / or conformational changes in membrane components that mediate endocytosis to enhance the endocytic efficacy and / or efficiency of the agent by preparing charged molecules or salt-forming techniques in the art, forming multivalent endocytic agents by linking charged, chargeable, and other hydrophilic chemical arms, or structurally modifying using medicinal chemistry strategies in the art, including the introduction of reversible or irreversible covalent bonds or moieties. The resulting endocytic agent can exist as a cationic or anionic agent in aqueous solution at a specific pH value and has both increased solubility and endocytic agent binding affinity and / or binding valence for picking up salt bridges, hydrogen bond interactions, or forming reversible / irreversible covalent bonds with membrane components that mediate endocytosis to enhance endocytic efficacy and / or efficiency. Moieties that can carry a charge or form hydrogen bonds, such as lysine, arginine, cysteine, serine, threonine, lipids, and carbohydrates, are often found in membrane components such as transmembrane proteins and tend to bind to ionic agents or agents with ionic bonds via salt bridges, hydrogen bonding interactions, or reversible covalent bond formation (Ondrej Kuda et al., Journal of Biological Chemistry, 288(22), 15547-15555, 2013; Dante Necukai et al., Nature, 504(7478), 172-176, 2013; Joanna SGSlusky et al., Bioinformatics, 29(17), 2122-2128, 2013; Yibo Wang et al., Journal of Physical Chemistry B, 125(8), 2124-2133, 2021; Anupam Bandyopadhyay et al., Current Opinion in Chemistry Biology, 34, 110-116, 2016). Thus, for example, by using reversible covalent chemical strategies known in the art for structural modification, including linking or replacing any chemical bond or moiety within the agent with a chemical bond or moiety that can form a reversible covalent bond with any nucleophile in biology (including lysine, arginine, cysteine, serine, threonine, and carbohydrates) (Anupam Bandyopadhyay et al., Current Opinion in Chemistry Biology, 34, 110-116, 2016), the resulting endocytosis agent can have increased binding affinity and / or binding valency to membrane components that mediate endocytosis, allow or increase dimerization or clustering of membrane components that mediate endocytosis, and / or allow or increase conformational changes of membrane components that mediate endocytosis, thereby enhancing endocytic efficacy and / or efficiency.Alternatively, by structural modifications, including linking or replacing any chemical bond or moiety within the agent with a chemical bond or moiety that can form a reversible or irreversible covalent bond with any nucleophile in biology, combined with strategies including linking or replacing any chemical bond or moiety within the agent with a cleavable chemical bond or moiety, the resulting endocytosis agent can have increased binding affinity and / or binding valency to the membrane component that mediates endocytosis, allow or increase dimerization or clustering of the membrane component that mediates endocytosis, and / or allow or increase conformational changes of the membrane component that mediates endocytosis to enhance endocytosis efficacy and / or efficiency, wherein the cleavable chemical bond or moiety in the resulting endocytosis agent can be cleaved in cells or in vivo, resulting in the release of the biologically active endocytosis agent from the membrane component that mediates endocytosis to exert any biological function. Therefore, the present technology allows endocytosis agents to spontaneously have increased solubility and / or permeability via endocytosis for disease diagnosis and treatment.

[0106] Another benefit of the present technology is that it allows for the adjustment of the metabolic stability of the endocytosis agent. In order to meet the stability requirements of the agent for treatment and diagnosis of a subject, the methods disclosed herein include, but are not limited to, adding or removing substituents, fragment replacement, cyclization, backbone transitions, bioisosteres, linkerology (linker-activity relationship studies), prodrugs, or reducing the overall LogP value. In certain embodiments, the methods refer to deuterium and / or fluorine replacement of any hydrogen atom on the endocytosis agent. In certain embodiments, the methods refer to the introduction of substitutes to reduce metabolism at soft spots in the agent. These methods allow for a balance to be achieved between the intended function of the endocytosis agent, the binding affinity to the membrane components that mediate endocytosis, and the metabolic stability for disease treatment.

[0107] In classical drug discovery and development activities, polar or hydrophilic compounds are determined to have poor permeability because high polarity or hydrophilicity makes it difficult for compounds to dissolve in the membrane lipid bilayer and diffuse through the cell membrane. As polarity increases, the permeability of polar or hydrophilic compounds drops sharply. However, proteins, especially membrane protein surfaces, usually contain hydrophilic amino acids facing the outside, and some post-translational modifications (PTM) parts (such as glycan groups) tend to be located on membrane protein surfaces that can be combined with polar compounds via salt bridges or hydrogen bonds, and the polar compounds include but are not limited to inorganic compounds, chelates, metal-based compounds and / or polar organic compounds, such as phosphatase inhibitors, peptide-based compounds, and membrane proteins often have small molecular binding areas. Therefore, one benefit of this technology is that endocytosis is a general approach to ingest endocytic agents, and there is no restriction on the molecular weight, polarity and lipophilicity of endocytic agents. In particular, one benefit of the present technology is that endocytosis can promote the cellular uptake of hydrophilic agents, including but not limited to inorganic compounds, chelates, metal-based compounds and / or polar organic compounds, such as phosphatase inhibitors and peptide-based compounds. For example, by appropriately linking a small molecule membrane protein binder to a phosphatase inhibitor or insulin, a salt bridge or hydrogen bond can be formed between the surface amino acids of the phosphatase inhibitor or insulin and the membrane protein to induce conformational changes during endocytosis. Using the present technology, polar compounds can be absorbed and used via oral administration. By introducing a facilitated diffusion glucose transporter (GLUT) binding portion onto a phosphatase inhibitor using a cleavable or non-cleavable bond or portion, the resulting endocytic phosphatase inhibitor agent can be used via oral administration due to increased binding affinity and / or binding valence to the membrane component that mediates endocytosis, and / or enhanced dimerization or clustering of the membrane component that mediates endocytosis, and / or enhanced conformational changes of the membrane component that mediates endocytosis for endocytosis. By introducing a CD36-binding moiety onto the insulin molecule using a cleavable or non-cleavable bond or moiety, the resulting endocytosed insulin agent can be administered orally due to increased binding affinity and / or valency to membrane components that mediate endocytosis, enhanced dimerization or clustering of membrane components that mediate endocytosis, and / or conformational changes in membrane components that mediate endocytosis for endocytosis. Insulin stability can be enhanced by exosome formation following endocytosis and endosomal escape.

[0108] Because the driving force for endocytic uptake of an agent is the binding between the endocytosed agent and the membrane components that mediate endocytosis, and because endocytosis is a process that can be independent of passive diffusion, one benefit of the present technology is that it can be used to allow endocytosed agents to cross cell membranes and / or body barriers via endocytosis alone or in combination with other mechanisms, including but not limited to passive diffusion, facilitated diffusion, transporter-mediated influx and / or efflux, and paracellular transport.

[0109] One benefit of this technology is that it expands the current concept of polypharmacology from seeking a combination or synergistic effect of the intrinsic activity of a drug on multiple disease-related targets or pathways (Rajan Chaudhari et al., Expert Opinion on Drug Discovery, 15(9), 1025-1044, 2020) to a combination or synergistic effect on disease-related targets or pathways and binding properties to membrane components that mediate endocytosis. The synergistic or additive effect on disease-related targets or pathways and membrane components that mediate endocytosis can reduce the requirement for the dose of the endocytosis agent for therapeutic purposes; because the increased binding affinity and / or binding valence of the endocytosis agent to the membrane components that mediate endocytosis, the dimerization or clustering of the membrane components that mediate endocytosis and / or the conformational change of the membrane components that mediate endocytosis can lead to enhanced endocytosis efficacy and / or efficiency, and better absorption via endocytosis, which can make up for the lack of intrinsic activity of some endocytosis agents on disease-related targets or pathways for disease diagnosis and treatment.

[0110] Because the binding properties of an endocytosis agent to a membrane component that mediates endocytosis can be independent of its ability to bind to an intrinsic pharmacological target, it allows the endocytosis agent to become a polypharmacological compound that spontaneously has binding affinity for both the membrane component that mediates endocytosis and the pharmacological target for intrinsic pharmacological activity. Therefore, another benefit of the present technology is a method for spontaneously identifying or generating new endocytosis agents via polypharmacological endocytosis and functional targeting. One method is to directly structurally modify the agent so that it is easy to bind to both the membrane component that mediates endocytosis and the pharmacological target for intrinsic pharmacological activity. Among them, the process of identifying or generating new polypharmacological endocytosis and functional targeting agents is used by classical medicinal chemistry in this field by spontaneously monitoring the activity of both endocytosis and biological target modulation efficiency. For example, to develop endocytic BCL-2 inhibitors, assays for CD36 binding affinity by SPR and BCL-2 targeting efficiency by SPR can be used in the classical medicinal chemistry optimization process in the art to identify or generate dual endocytic and BCL-2 targeting agents. Multiple chemical arms in the ligand help improve binding affinity for both CD36 and BCL2, and / or efficient uptake. In addition, multivalent agents or agents with increased binding valence can enhance dimerization or clustering of membrane components and / or induce conformational changes in membrane components to activate the endocytic cascade. In addition, as revealed by crystallographic studies and molecular modeling, bell-shaped ligands or macroligands containing multiple side chains can produce good alignment with the binding pocket of CD36 (residues 127-279). Therefore, another approach disclosed herein for identifying or generating novel endocytic agents with enhanced multiple pharmacological properties, including endocytic efficacy and / or efficiency and targeted biological functions, is to simply attach or fuse additional chemical arms with binding affinity for membrane components mediating endocytosis and / or target proteins to the endocytic agent via surface or intra-pocket interactions via cleavable or non-cleavable chemical bonds or linker units to make them multivalent compounds. The resulting multivalent endocytic agent can 1) have increased affinity and / or binding valency for membrane components mediating endocytosis, and / or increased activity against biological targets, 2) allow or increase dimerization or clustering of membrane components mediating endocytosis, or 3) allow or increase conformational changes in membrane components mediating endocytosis to enhance endocytic efficacy and / or efficiency and be better absorbed via endocytosis, while still retaining or increasing targeted biological functions. Chemical arms with affinity for CD36 and biological targets can be constructed by library synthesis and subsequent screening. Compared to existing approaches in medicinal chemistry that adjust one intrinsic property of an agent via structural modification but inevitably affect other properties, this technology allows increasing the permeability of endocytic agents without sacrificing or even enhancing solubility, stability and their intrinsic pharmacological activity for use in disease research, diagnosis, prevention and treatment.

[0111] As a result of endocytosis, foreign substances can be picked up by cells and released from (donor) cells in the form of extracellular vesicles (EVs) via exocytosis or membrane fusion, including but not limited to exosomes, apoptotic bodies and microvesicles (MVs) or extranuclear granules (ectosomes), wherein foreign substances can be present at any site of the extracellular vesicles. Endogenous extracellular vesicles can be taken up by any (recipient) cell via endocytosis, membrane fusion or endocytosis, resulting in foreign substances being transported across more than one cell layer (Ravi Shah et al., The New England Journal of Medicine, 8 (379), 958-966, 2018; Oscar PB Wiklander et al., Science Translational Medicine, 11 (492), eaav8521, 2019; Raghu Kalluri et al., Science, 367 (6478), eaau6977, 2020). Thus, another benefit of the present technology is a method for transporting endocytic agents across more than one cell layer in the body, wherein the endocytic agent can be taken up by cells via endocytosis and can be released in the form of free endocytic agent molecules and / or endocytic agent-vesicle complexes (i.e., exocytic vesicles), wherein the complex comprises the endocytic agent and a lipid bilayer vesicle, including an extracellular vesicle, and the exocytic vesicle can be taken up by any (receptor) cell via endocytosis or membrane fusion to act for any application purpose. In particular, an additional benefit of the present technology is a "one-step" method for generating and using exocytic vesicles in humans and animals, wherein, without further processes including agent or vesicle isolation, cells in the body pick up the endocytic agent via endocytosis and then secrete the exocytic vesicle via exocytosis, and the resulting endogenous exocytic vesicles can be directly distributed to any tissue and used by the body, including the brain, for any purpose.

[0112] Another benefit of the present technology is a method for preparing, isolating and using exocytic vesicles for any purpose in vitro and / or in vivo. In particular, one method for generating exocytic vesicles is that cells in an animal body can take up an endocytic agent via endocytosis and then spontaneously secrete exocytic vesicles via exocytosis. Another method for generating functional exocytic vesicles is that the endocytic agent molecule can be linked to extracellular vesicles and / or exocytic vesicles in situ via covalent or non-covalent bonds. Another method for generating exocytic vesicles is to load an endocytic agent into an exocytic vesicle in vitro by any technique in the art. Another method is to isolate exocytic vesicles from cells, body fluids, tissues, organs, products or culture media by any extracellular vesicle isolation technique in the art.

[0113] Because extracellular vesicles (EVs) carry soluble substances, such as soluble cytokines, EVs are allowed to have sufficient solubility in aqueous solutions (Ana Paula Ramos et al., Journal of Extracellular Biology, 1(1), e34, 2022). Therefore, another benefit of this technology is a method for preparing and using exocytic vesicles for any purpose in vitro or in vivo to increase the water solubility of endocytic agents. For example, in order to have water solubility in an animal, a method for increasing the water solubility of an agent is to allow or increase the uptake of an endocytic agent via the above method, resulting in an increase in the loading of the endocytic agent into the exocytic vesicle and / or the release of the exocytic vesicle via the endocytic / exocytic process. The endocytic agent can be dissolved in an aqueous solution in the form of exocytic vesicles.

[0114] Another benefit of the present technology is a method for adjusting the absorption, distribution, metabolism and excretion (ADME) properties of endocytosed agents in animals. For example, in order to prolong the residence time and / or half-life of a given endocytosed agent in an animal, the method of the present technology is to allow or increase the uptake of the endocytosed agent by endocytosis via the above method, so as to allow or increase the loading of the endocytosed agent into exocytic vesicles via endocytosis and the release of exocytic vesicles, for reducing or preventing the first-pass metabolism and / or excretion of the endocytosed agent. In order to improve the brain permeability of the agent, the method of the present technology is to allow or increase the uptake of the endocytosed agent by endocytosis via the above method, so as to allow or increase the loading of the endocytosed agent into exocytic vesicles via endocytosis and the release of exocytic vesicles, for reducing or preventing the blood-brain barrier efflux transport of the endocytosed agent.

[0115] Another benefit of the present technology is a method for evaluating and / or determining the ADME properties of endocytic agents during drug discovery and development, which includes a process or step for isolating and / or lysing exocytic vesicles from a sample. For example, in certain embodiments, the concentration of endocytic agent in the blood or tissue after administration of the endocytic agent includes free endocytic agent molecules in the blood or tissue and endocytic agent molecules embedded in exocytic vesicles (such as exosomes) in the blood or tissue. In order to release the endocytic agent from the exocytic vesicle for endocytic agent detection, any technology and skills used in the art for isolating and / or lysing EVs are applicable to isolating and / or lysing the exocytic vesicles of the present disclosure.

[0116] Another benefit of the present technology is a method for reducing the toxicity of an agent / drug by preparing and using exocytic vesicles containing an endocytic agent in vitro or in vivo. Hematological toxicity represents the main toxicity of cytotoxic agents (Etienne Chatelut et al., Investigational New Drugs, 21, 141-148, 2003). For example, most PARP inhibitors in the clinic are observed to have hematological toxicity, including neutropenia, anemia, thrombocytopenia, fatigue and bleeding, which is due to the toxicity of PARP inhibitors to platelets, leukocytes and erythrocytes (Yamin Shu et al., Cancer Medicine, 12(3), 3365-3375, 2023). As disclosed in the present disclosure, the endocytic agent can be taken up by cells and released from the cells in the form of exocytic vesicles (such as exosomes loaded with the endocytic agent), wherein the endocytic agent can be present in the exosomes, preventing or reducing direct contact of free endocytic agent molecules with blood cells. In addition, as natural particles, exosomes have unique properties, such as inherent stability and low immunogenicity (Raghu Kalluri et al., Science, 367, eaau6977, 2020). Therefore, another benefit of this technology is the use of membrane components in cells and tissues that mediate endocytosis and the exocytosis process to release exocytic vesicles to reduce drug toxicity by preventing or reducing the toxicity of the agent / drug. For example, after oral administration of an endocytic agent, cells in the gastrointestinal tract can pick up the endocytic agent via endocytosis and then secrete exocytic vesicles into the circulation system, and the resulting exocytic vesicles reduce hematological toxicity compared to free endocytic agent molecules (Ashish K. Agrawal et al., Nanomedicine, 13, 1627-1636, 2017). In particular, an additional benefit of the present technology is a method for reducing the toxicity of an agent by adjusting the endocytic efficacy and / or efficiency through any of the structural modification methods disclosed in the present technology, loading the endocytic agent into exocytic vesicles and / or releasing exocytic vesicles in different cells and tissues, allowing endocytosis or adjusting the endocytic efficacy and / or efficiency.

[0117] Another benefit of this technology is that different expression of membrane components that mediate endocytosis in cells and tissues can be used to increase the effectiveness of endocytic agents and reduce their toxicity. Changes in metabolic activity have been shown to support the malignant nature of cancer cells (Ralph J. DeBeradinis et al., Science Advances, 2 (5), e1600200, 2016). For example, recent studies have confirmed that high CD36 expression plays a key role in tumor initiation, development, invasion and metastasis by providing tumor cells with an increased supply of nutrients (such as fatty acids and lipids) (Gloria Pascual et al., Nature, 541, 41-45, 2017; Matthew J. Watt et al., Science Translational Medicine, 11 (478), eaau5758, 2019). Therefore, by adopting the overexpression of membrane components that mediate endocytosis on cancer cells or the nutrient uptake that is upregulated by endocytosis in cancer cells to target the delivery of drugs, the potential toxicity to normal cells can be minimized and the therapeutic window of the drug can be expanded. Although the examples in the present disclosure demonstrate that CD36-mediated endocytosis mediates the uptake of endocytic agents into cells, other membrane components that mediate endocytosis, such as scavenger receptors (SR), endothelial cell protein C receptor (EPCR), fatty acid binding protein (FABPpm), fatty acid transporter protein (FATP), free fatty acid receptor 1 (GPR40) and epidermal growth factor receptor (EGFR), facilitated glucose transporter (GLUT), including all variants, mutations, splice variants, insertions and deletions and fusions, described in but not limited to (Sara Sigismund et al., Nature Review Molecular Cell Biology, 22, 625-643, 2021), can also be used to take up agents via endocytosis. In addition, other chemical modifications to endocytic agents that provide stronger binding affinity or greater binding valency to membrane components mediating endocytosis, allow or increase dimerization or clustering of membrane components mediating endocytosis, and / or allow or increase conformational changes in membrane components mediating endocytosis to enhance endocytic efficacy and / or efficiency can serve as a platform for enhanced efficacy and / or efficiency of uptake of drugs, particularly eRo5 and bRo5. Thus, differential expression of endocytic membrane proteins can be used for patient stratification of endocytic drugs.

[0118] Another benefit of the present technology is that the different expression of membrane components that mediate endocytosis in cells and tissues can be used for special delivery systems, such as topical, inhalation, intraperitoneal, intravenous, microinjection and oral delivery. For example, due to the abundant expression of membrane components that mediate endocytosis (such as CD36 or GLUT) on the luminal surface of enterocytes in the intestine (Fatiha Nassir et al., Journal of Biological Chemistry, 282 (27), 19493-19501, 2007), by appropriately optimizing the affinity and metabolic stability of membrane components that mediate endocytosis, endocytic agents, especially those located in the chemical space of eRo5 and bRo5, will have acceptable oral bioavailability. Another benefit of the present technology is that endocytic agents can be administered by any appropriate route, including oral (including buccal or sublingual), topical (including buccal, sublingual, transdermal or eye drops), inhalation, parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes, intraperitoneal injection and microneedle patches.

[0119] Another benefit of this technology is that the conformational diversity of the membrane components that mediate endocytosis can be used for the structural design and modification of endocytic agents. Membrane proteins have various conformational states in different cells or under different conditions, including but not limited to cell size and volume, cell microenvironment and post-translational modification state (Raghavendar Reddy SangannaGari et al., Nature Communications, 12, 4363, 2021; Diego del Alamo et al., eLife, 11, e75751, 2022; Alex R Terry et al., Cell Metabolism, 35, 1-7, 2023). The different conformations of membrane proteins show unique sensitivity to ligands (Dante Necukai et al., Nature, 504 (7478), 172-176, 2013; Fu-Lien Hsieh et al., Nature Communications, 7, 12837, 2016). Therefore, one benefit of the present technology is the ability to design or structurally modify endocytic agents for any purpose using medicinal chemistry strategies known in the art, or to target specific conformations of membrane components that mediate endocytosis. For example, the CD36 protein can adopt different conformations on the membranes of normal cells and cancer cells. It is reasonable to exploit the conformational diversity of CD36 on normal cells and cancer cells to design or modify endocytic agents using medicinal chemistry strategies known in the art, or to select endocytic agents to target specific conformations of CD36 on cancer cells to enhance anti-tumor efficacy and reduce toxicity. Since protein conformational transitions change their shape and biological function upon receiving input signals (such as ligand binding, chemical modification or environmental changes) (Matin Dutertre et al., Perspective in Pharmacology, 295(2), 431-437, 2000), one benefit of the present technology is that any method in the art (such as applying mechanical, electrical, thermal, cold, light or radiation stimulation and / or the presence of an endocytosis agent) can be used to change the conformation of membrane components that mediate endocytosis, resulting in changes in biological events in the cell and / or changes in the sensitivity of membrane components that mediate endocytosis to endocytosis agents.

[0120] Another benefit of this technology is that the differential expression and / or conformation of membrane components that mediate endocytosis can be used to provide precise health care for individuals using this endocytosis agent. At the same time, feedback from clinical endocytosis therapy can be used to adjust the administration route of the endocytosis agent and / or improve the results of endocytosis therapy. Studies have shown that there is differential expression of membrane components between individuals, between cells and tissues within an individual, and between different disease stages within an individual (Roy L. Silverstein et al., Science Signaling, 2(72), re3, 2009; Aritro Nath et al., Scientific Reports, 4(6), 18669, 2016; Matthew J. Watt et al., Science Translational Medicine, 11(478), eaau5758, 2019). In addition, membrane proteins have various conformational states and / or post-translational states on different cells or under different conditions, resulting in different sensitivities of membrane proteins to ligands. The present disclosure demonstrates that endocytosis is a cellular uptake pathway for endocytic agents, and that membrane components that mediate endocytosis (such as CD36) have different expression and / or structural conformations in cells and / or tissues of different patients (Vincenza Cifarelli et al., Comprehensive Physiology, (8) 2, 493-507, 2018; Hélène Poirier et al., European Journal of Biochemistry, 238 (2), 368-373, 1996). Therefore, one benefit of the present technology is to stratify patients to personalize the selection of endocytic agents and routes of administration of endocytic agents, wherein membrane components that mediate endocytosis can be used as biomarkers in cells and / or tissues, and the different expression and / or conformations of membrane components that mediate endocytosis in patient cells and / or tissues are used to select and / or administer endocytic agents to treat subjects in terms of patient stratification, route of administration, and dose selection. The diversity of genetic profiling is widely present between different cells, tissues, and the body. It is reasonable to exploit the differences in genetic maps between cells, tissues, and the body to drive bioactive agents in the drug discovery and development value chain. As the first PROTAC targeting the androgen receptor (AR) to enter clinical trials, although ARV-110 was able to completely degrade AR and most of its point mutants in preclinical studies, it was found in clinical studies that tumor patients carrying ART878 or H875 point mutations were particularly sensitive to ARV110 treatment (Xin Gao et al., Journal of Clinical Oncology, 40(6,) suppl.017, 2022).Since we have demonstrated that all of these agents / drugs / vesicles act as endocytic agents, including but not limited to the agents disclosed in the Examples herein (Table 1), precise selection of patients with high expression and / or ligand-sensitive conformations of membrane components mediating endocytosis (such as CD36) in diseased tissues and / or absorptive tissues (such as the intestine) can increase the clinical benefit of endocytic agents to patients.

[0121] Another benefit of this technology is that endocytic agents can be used to study, diagnose, prevent and treat any subject, including but not limited to aging and age-related diseases and conditions, weight management, cancer, central nervous system (CNS) diseases and conditions, cardiovascular disease (CVD), diabetes, eye diseases and conditions, hypertension, immune system diseases, infections, inflammation and inflammation-related diseases and conditions, obesity and obesity-related diseases and conditions, respiratory diseases and conditions, and skin diseases and conditions. Drug discovery for treating subjects, especially CNS or skin diseases and conditions, has been challenging due to the body's barrier to foreign substances (Sung Min Pyo et al., Skin Pharmacology and Physiology, 32, 283-293, 2019; William M. Pardridge, NeuroRX, 2(1), 3-14, 2005). Endocytosis and exocytosis result in the release of extracellular vesicles (EVs) and free nutrients through the process of exocytosis (Marcel Grapp et al., Nature Communications, 4, 2123, 2013; Raghu Kalluri et al., Science, 367(640), 2020), which facilitates the penetration of nutrients and / or EVs in free form across membrane barriers such as the blood-retinal barrier (Mónica Díaz-Coránguez et al., Vision Research, 139, 123-137, 2017), lung endothelial and epithelial barriers (Mikihisa Takano et al., Expert Opinion on Drug Delivery, 12(5), 813-825, 2015; Julia Voigt et al., Proceedings of the National Academy of Sciences, 111(8), 2942-2947, 2014), skin barrier (Noriaki Nagai et al., International Journal of Molecular Biology, 2014), and the skin barrier (Mónica Díaz-Coránguez et al., Vision Research, 139, 123-137, 2017). Sciences, 19(7), 2138, 2018) and brain-blood barrier (Mathew W. Smith et al., Journal of Drug Targeting, 14(4), 191-214, 2006).For example, by binding to receptors or proteins expressed in the blood-brain barrier (such as scavenger receptors (e.g., CD36), major facilitator superfamily domain-containing protein 2 (Mfsd2a), flotillin-1, flotillin-2, glucose transporter 1 (GLUT1), glutathione transporter, amino acid transporter (e.g., L-type amino acid transporter 1, LAT1), transferrin receptor, lactoferrin receptor, low-density lipoprotein receptor, nicotinic acetylcholine receptor, insulin receptor, insulin-like growth factor receptor, integrin (e.g., αVβ3 integrin) and / or CD13 / APN receptor), endocytic agents can be taken up into brain endothelial cells, cross the brain-blood barrier via endocytosis, and reach the brain parenchyma via the endocytic / exocytic pathway in the form of free endocytic agents, exocytic vesicles, or a mixture of free endocytic agents and exocytic vesicles. In addition, due to endocytosis, endocytic agents are taken up into the cell and located in intracellular organelles and / or vesicles (such as endosomes and multivesicular bodies), thereby reducing or preventing efflux transporters (such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins MRP1, MRP3, MRP4, and MRP6) from pumping the endocytic agent out of the cell. It should be understood that the expression of membrane components that mediate endocytosis in membrane barriers for any purpose and / or any structural modification of the agents disclosed herein can enhance the efficacy and / or efficiency of endocytic agent endocytosis and enhance the transport of endocytic agents across membrane barriers. Therefore, a particular benefit of the present technology is that endocytic agents can be used to study, diagnose, prevent and treat ocular, respiratory, skin and CNS conditions and diseases because, following membrane component-mediated endocytosis, endocytic agents can be transported across membrane barriers via the endocytic / exocytic pathway in the form of free endocytic agents, exocytic vesicles, or a mixture of free endocytic agents and exocytic vesicles in any ratio.

[0122] Another benefit of the present technology is a method for identifying bioactive compounds known in the literature that can be taken up by cells via endocytosis. The method comprises adjusting (such as increasing, decreasing or deleting) the expression of membrane components that mediate endocytosis in cells, tissues and / or bodies by editing, knocking down or silencing the membrane components that mediate endocytosis, and then comparing the activity of the bioactive compound in cells, tissues and / or bodies with and without editing, knocking down or silencing the membrane components that mediate endocytosis. The compound taken up via endocytosis has significantly increased or decreased biological activity in cells, tissues and / or bodies that have membrane components that mediate endocytosis, knocking down or silencing, compared to the activity of the compound in cells, tissues and / or bodies that do not have membrane components that mediate endocytosis, knocking down or silencing the membrane components that mediate endocytosis.

[0123] Another benefit of the present technology is a method for identifying biological targets in cells and the body using identified endocytic agents as probes by any technology and skill in the art. For example, the identified endocytic agents having biotin, fluorescence, Halo-tag ligands, SNAP-tag ligands, CLIP-tag ligands, or chemical bonds or moieties that can form covalent bonds with any biological target are particularly suitable for use with microscopic imaging, immunophenotyping, immunoprecipitation, flow cytometry, fluorescence activated cell sorting (FACS), fluorescence resonance energy transfer (FRET) and / or omics (including genomics, epigenomics, transcriptomics, proteomics and metabolomics) analysis to identify biological targets of endocytic agents and derivatives. In particular, one benefit of the present technology is a method for identifying membrane targets that mediate endocytosis. A method for identifying membrane targets that mediate endocytosis can use a labeled endocytic agent followed by administration of an agent. Detection and identification techniques include microscopic imaging, immunoprecipitation, immunophenotyping, flow cytometry, fluorescence activated cell sorting (FACS), fluorescence resonance energy transfer (FRET), omics (including genomics, epigenomics, transcriptomics, proteomics and metabolomics) analysis and / or positron emission tomography (PET) scanning using computed tomography (CT) or magnetic resonance imaging (MRI). For example, a labeled probe (such as biotin) is attached to any appropriate site of the endocytosis agent to produce a biotin-labeled endocytosis agent. Subsequently, the endocytosis of membrane components that mediate endocytosis of the targeted endocytosis agent can be identified by culturing the cells with the biotin-labeled endocytosis agent, isolating the membrane proteins, and then performing immunoprecipitation, FACS, omics analysis, and protein blot confirmation. Another method is to use any genome scanning technology in the art to identify membrane targets that mediate endocytosis. For example, comparison of gene expression between cells, tissues, or bodies with different sensitivities to certain endocytosis agents can be used to identify membrane targets that mediate endocytosis. Another approach is to use any gene editing technology in the art to identify membrane targets that mediate endocytosis, where gene-edited cells, tissues, or bodies can be more sensitive or more resistant to endocytosis treatment. For example, cells whose gene expression is suppressed and activated by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa), respectively, respond differently or complementary to certain endocytosis treatments, which can be used to identify membrane targets that mediate endocytosis.

[0124] As used herein, "gene editing" refers to the process of artificially introducing genetic modifications. Genetic engineering can be performed at the DNA, RNA or epigenetic level. Genetic modification includes: (i) deleting endogenous genes; (ii) introducing recombinant nucleic acids encoding wild-type or mutant forms of endogenous or exogenous proteins; (iii) introducing RNA molecules (e.g., small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA and microRNA (miRNA)) that interfere with the functional expression of proteins; or (iv) changing the promoter or enhancer elements (i.e., regulatory elements) of one or more endogenous genes. It should be understood that item (ii) includes replacing endogenous genes with genes encoding altered or completely different proteins (e.g., by homologous recombination), and item (iv) includes modifying or manipulating the regulatory region of the target gene or any region adjacent to the target gene (e.g., up to 5KB on either side of the target sequence). Genetic engineering also includes changing endogenous genes to produce proteins with additions (e.g., heterologous sequences), deletions or substitutions (e.g., mutations, such as point mutations; conservative or non-conservative mutations). Mutations can be introduced specifically (e.g., by site-directed mutagenesis or homologous recombination) or can be introduced randomly (e.g., chemical mutagenesis). Thus, genetic modification can regulate genes in several ways, such as increasing expression, increasing function, decreasing expression, decreasing function, or gene knockout. Exemplary methods include, but are not limited to, RNA-based RNA interference, including small interfering RNA (siRNA) and short hairpin RNA (shRNA), DNA-based RNA interference, including antisense oligonucleotides, and CRISP-mediated genome editing technology.

[0125] Another benefit of this technology is that when the present endocytic agent is used or selected for any purpose, different microenvironmental factors of cells and tissues can be used as modulators, where the microenvironment of cells and tissues includes but is not limited to pH, salinity, oxygen gradient, carbon dioxide gradient, H2O2 gradient, nutrient gradient, and therapeutic compound gradient. For example, due to the acidic environment of cancer cells, endocytic agents with basic groups can be used for tumor-targeted delivery to enhance therapeutic efficacy and reduce toxicity. Similarly, endocytic agents with basic amino groups can be used to enhance BBB (blood-brain barrier) localization to obtain higher therapeutic efficacy and lower toxicity.

[0126] Another benefit of this technology is that when the present endocytosis agent is used or selected for any purpose, expressions of membrane components that mediate endocytosis with different conformations, isoforms (or variants) and / or post-translational modifications can be used as modulators. Another benefit of this technology is that when the endocytosis agent of the present invention is used or selected for any purpose, expressions of cofactors that form complexes with membrane components that mediate endocytosis can be used as modulators. Membrane proteins have different conformations in different environments, and proteins with different conformations exhibit different affinities for ligands or other proteins and biological functions. Membrane protein isoforms can have unique expression differences between cells and individuals and / or functions. In addition, the diversity of glycosylation states in membrane proteins can produce complex pleiotropy, where a unique modification on one glycosylation site may alter function or recognition within a specific cellular environment, but may cause other effects or be functionally silent in other environments. Therefore, for example, by targeting the specific conformation, isoform, glycosylation, palmitoylation or phosphorylation state of membrane components or cofactors that mediate endocytosis in cancer cells, endocytosis agents can be used to enhance therapeutic function and reduce toxicity in cancer treatment.

[0127] Another benefit of this technology is that the endocytosis agent binds to the membrane components that mediate endocytosis and triggers the internalization of the membrane via the endosomal / lysosomal pathway for digestion. This technology can be used to deliver disease-related membrane components or extracellular substances that mediate endocytosis into cells via endocytosis using an endocytosis agent, and destroy or degrade disease-related membrane components or extracellular substances that mediate endocytosis via the endosomal / lysosomal system. Disease-related membrane components or extracellular substances that mediate endocytosis include, but are not limited to, membrane proteins or extracellular proteins, carbohydrates, lipids, pathogens, particles, viruses, fungi, protozoa, bacteria, vectors, cell debris, and another cell.

[0128] Another benefit of the present technology is that it allows for activation of the endocytic process or increase in expression of endocytic-mediated membrane components via regulatory input signals (such as binding of endogenous or exogenous substances to membrane proteins) to enhance endocytic absorption of endocytic agents. For example, the binding of insulin to the insulin receptor on the membrane activates the endocytic cycle of membrane components that mediate endocytosis and / or increases the expression and / or relocation of membrane components that mediate endocytosis (such as GLUT), resulting in enhanced absorption of endocytic agents via endocytosis. It is understood that endocytic agents can simultaneously bind to membrane components that mediate endocytosis and membrane components that do not mediate endocytosis to activate the endocytic process or increase the expression of membrane components that mediate endocytosis and enhance absorption via endocytosis.

[0129] like Figure 1A As shown in , disclosed herein is an endocytosis agent or a salt thereof, which can bind to a membrane component that mediates endocytosis and be taken up into cells via endocytosis. Figure 1AAlso disclosed are methods for drug discovery that allow or adjust the binding affinity and / or valency of an agent to a membrane component that mediates endocytosis to enhance endocytic efficacy and / or efficiency.

[0130] exist Figure 1B In the present invention, the absorption mechanism and subsequent distribution, metabolism and excretion (ADME) of endocytosed agents in different administration routes (including oral, topical and inhaled) are disclosed. Also disclosed are methods for evaluating or determining the ADME properties of endocytosed agents during drug discovery and development.

[0131] like Figure 1C As shown in, methods for the study, diagnosis, prevention and treatment of any subject, especially for CNS conditions and diseases, by administering endocytic agents or exocytic vesicles via oral or intravenous routes are disclosed. Also disclosed are methods for the study, diagnosis, prevention and treatment of any disease by combining endocytic agents with extracellular vesicles (EVs).

[0132] like Figure 1D As shown in , the mechanism of action of the endocytic agent is disclosed. The absorbed endocytic agent or extracellular vesicle, or the exocytic vesicle containing the endocytic agent, can be absorbed into the cell via endocytosis or membrane fusion for any application purpose to exert its effect.

[0133] Also disclosed are methods for generating and using exocytic vesicles in humans or animals using a "one-step" approach, wherein, without further steps, including isolation of the agent or vesicles, cells in vivo take up an endocytic agent via endocytosis and subsequently secrete the exocytic vesicle, and the resulting endogenous exocytic vesicles can be directly used by the body for any purpose. Also disclosed are methods for isolating exocytic vesicles. One method for preparing exocytic vesicles is for cells to take up an endocytic agent via endocytosis and then secrete the exocytic vesicle via exocytosis. Another method for forming functional exocytic vesicles is for the endocytic agent molecule to be linked in situ to extracellular vesicles and / or exocytic vesicles via covalent or non-covalent bonds. Methods for preparing and using the same are also disclosed.

[0134] Endocytotic agents

[0135] An endocytic agent or pharmaceutical agent refers to any compound or portion thereof or conjugate that can bind to a membrane component that mediates endocytosis and be taken up by the cell via endocytosis. In some embodiments, the pharmaceutical agent is a therapeutic agent, a diagnostic agent, a binding agent, a conjugate, a nanoparticle, or a vesicle. A therapeutic agent is any compound that can be used to treat a subject. A diagnostic agent is any compound that can be used to provide qualitative or quantitative information about a biomolecular target of interest or a biological environment. A binding agent is any compound that binds to a membrane component that mediates endocytosis or extracellular material, including membrane proteins or extracellular proteins, carbohydrates, lipids, pathogens, particles, viruses, bacteria, fungi, protozoa, vectors, cell debris, and cells. A conjugate is a compound composed of one or more pharmaceutical agents conjugated to one or more chemical arms via a cleavable or non-cleavable chemical bond or linker unit. A vesicle is an intracellular or extracellular structure composed of a liquid or cytoplasm surrounded by a lipid bilayer.

[0136] As discussed below, the endocytic agents of the present disclosure include, but are not limited to, neutral compounds, free bases or acids, their salts, solvates, and prodrugs, and may include oxidized sulfur atoms or quaternized nitrogen atoms in their structures, even if not explicitly described or shown, particularly pharmaceutically acceptable forms thereof. Such forms, particularly pharmaceutically acceptable forms, are intended to be encompassed by the appended claims.

[0137] In some embodiments, the binding affinity of the endocytosis agent to the membrane component that mediates endocytosis is K D The value was lower than 20.0 mM.

[0138] In some embodiments, the molecular weight of the endocytosis agent is greater than 200 Da. In certain embodiments, the molecular weight of the endocytosis agent is greater than 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1000 Da, 1100 Da, 1200 Da, 1300 Da, 1400 Da, 1500 Da, 1600 Da, 1700 Da, 1800 Da, 1900 Da, 2000 Da, 2100 Da, 2200 Da, 2300 Da, 2400 Da, 2500 Da, 2600 Da, 2700 Da, 2800 Da, 2900 Da, 3000 Da. In some embodiments, the molecular weight of the endocytosis agent may be between 200 and 10,000 Da.

[0139] In some embodiments, the endocytosis agent is a therapeutic agent. Exemplary therapeutic agents include, but are not limited to, drugs, protein inhibitors or antagonists, protein activators or agonists, protein modulators, molecular glues that induce or stabilize protein-protein interactions, protein degraders or multivalent agents, protein binders, diagnostic agents or chemical probes or vesicles, including their isotopomers, such as deuterium and / or fluorine-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers. Therapeutic agents are also protein, DNA or RNA modifying agents.

[0140] In certain embodiments, the therapeutic agent is a drug that can bind to a membrane receptor involved in the endocytic process. Exemplary drugs include, but are not limited to, any of the following compounds: 68Ga-DOTA-FAPI-46, 68Ga-DOTA-2P9FAPI)2, ABT-737, acarbose, acetaminosalol, actinomycin D, adenosine A1 receptor (A1R) agonists, aliskiren, AMG-131, argatroban, ascomycin, asukamycin, asunaprevir, atazanavir, atorvastatin, AZD5153, azithromycin, B-cell lymphoma 2 (BCL-2) Family protein inhibitors (such as venetoclax (ABT-119), navitoclax (ABT-263), APG-2575 (Lisaftoclax), UBX1325 (CAS No.: 2271269-01-1), APG-1252 (Pelcitoclax), APG-2575 (Lisaftoclax), AMG176, AMG397, AZD4606 (CAS No.: 22 41039-81-4), AZD5991, AZD4747 (CAS: 2489226-14-2), BGJ-398, birinapant, bis(7)-tacrine, BLU-945, BMS-777607, BMS-791325, BMS-986165 (CAS: 1609392-27-9), and brigatinib (CAS: 1197953-54-0 ), Brigimadlin, camdronate, camptothecin, candoxatril, capreomycin, ceritinib, CHF-6366, clarithromycin, cobimetinib, combretatropone, compound 28 (Fedor Romanov-Michailidis et al., Journal of Medicinal Chemistry, 66, 6122-6148, 2023), cosmomycin DD), CPT-Gly-PEG-folic acid, CUDC-101, CUDC-907, cyclosporine A, daclatasvir, dactinomycin, dalfopristin, danamide F, danaprevir, Danoprevir (CAS: 850876-88-9), Danuglipron (CAS: 2230198-02-2), DHP1808, diaspirine, digoxin, diprovocim-X, divarisib (CAS: 241798 7-45-0), diamino allose phosphate (DAP), DNL343, doxorubicin, dasatinib, DU1301, ECPU-0001, EDO-S101, EML981, epcoritamab (CAS: 2134641-34-0), eptifibatide, EPZ-5676, ergotamine, Eritoran, erythromycin A, erythronolide, etoposide, estramustine, ethacraplatin, everolimus, EZN-2208 (CAS: 946062-05-1), FAPI-46, FAPI-dimer, Fenebrutinib (CAS: 1434048-34-6), fedratinib, fosinopril (CAS: 98048-97-6), fostamatinib, G protein-coupled receptor (GPCR) inhibitor, G protein-coupled receptor (GPCR) agonist, gartisertib, Glecaprevir (CAS: 1365970-03-1), himeic acidA), histone deacetylase (HDAC) inhibitors, homoharringtonine, inhibitor of apoptosis (IAP) protein inhibitors (such as AZD5582 (CAS: 1258392-53-8), SM-164 (CAS No.: 957135-43-2) and xevinapant), indoleamine 2,3-dioxygenase (IDO) inhibitors, indobufen, IR820-SS-CPT, IT-101, itraconazole, ivermectin, JNJ78394355, JS230, KX2-361, ladostinigil, lapatinib, leucomycin ), Lipitor (atorvastatin), lonafarnib, Lumakras (CAS: 2296729-00-3), LUNA18 (CAS: 2676177-63-0), LY3502970 (CAS: 2212020-52-3), mammalian target of rapamycin (mTOR) and / or FK506 binding protein (FKBP) protein inhibitors, medoxomil, Milademetan (CAS: 1398568-47-2), MIP-1404, MK-1468, MK-8768 (CAS: 1432729-22-0), mometasone furoatefuroate), motixafortide (CAS: 664334-36-5), MPI8 (CAS: 856242-63-2), MRT-2359 (CAS: 2803881-11-8), MRTX-849 (CAS: 2326521-71-3), Muvalaplin (CAS: 2565656-70-2), NDI-034858 (CAS: 227290 4-53-5), nilotinib, nintedanib, NOSH-aspirin (NBS-1120), NKTR-102 (CAS: 1193151-09-5), NKTR-105, nirmatrelvir, ODDA-PTX, omaveloxolone, anobinost, orta taxel), ouabain, PAANIB-1, paclitaxel, Pacritinib (CAS: 937272-79-2), pelabresib, Pevonedistat (CAS: 905579-51-3), PF-03715455, phakellistatin, pictilisib, Piflufolastat F-18 injection, Pluvicto (CAS No.: 1703749-62-5), peroxisome proliferator-activated receptor (PPAR) agonists, PRMT inhibitors, protein phosphatase inhibitors, protein arginine methyltransferase (PRMT) inhibitors, pralsetinib, pseudomonic acid A acid A), PSMA-11, PSMA-617, PSMA-1007, PSMA I&S, PSMA I&T, PSMA SMOL-TTC monomer, PSMA SMOL-TTC dimer, PSMA SMOL-TTC trimer, PSMASMOL-TTC tetramer, PU-H71, pyrilutamide, quizartinib (CAS: 950769-58-1), quinupristin, rapamycin, Rapalink-1, REC-3599, rifampicin, rifapentine, rifabutin, rifaximin, Rilzabrutinib ib)(CAS:1575596-29-0), ritonavir, rivaroxaban, RMC-4998(CAS:2642037-07-6), RMC-6291(CAS:2641998-63-0), RMC-6236(CAS:2765081-21-6), roxithromycin, RPT193(CAS:2366152-15-8), sanguinamide A A), S63845, S64315, saquinavir, scavenger receptor (SR) inhibitors, activators or binders, endothelial protein C receptor (EPCR) inhibitors, activators or binders, setileuton, SHP-1971, simeprevir, sirolimus, solute carrier (SLC) transporter inhibitors, activators or binders, simeprevir (CAS: 923604-59-5), sparsentan (CAS: 254740-64-2), spiramycin, staurosporine, interferon-based STING agonists, Sug-HisVal-CPT, tacrolimus, taladegib, tapotoclax (CAS: 1883727-34-1), Tat-P4-(C5)2 (doi.org / 10.15252 / emmm.201911248), TNG348, tryptophan 2,3-dioxygenase (TDO) inhibitors, telithromycin, thiosptrepton, tinostamustine, tirbanibulin (KX2-391), triamcinolone phosphatase inhibitors, thiostriptyline, tinostamustine, tirbanibulin (KX2-391), triamcinolone phosphatase inhibitors, tiosptrepton, tinostamustine, tiospiriol phosphatase inhibitors ... trioxaquine, trypdronate, tubacin, tubocurarine, VCP746 (CAS No.: 1582751-84-5), volasertib, vazegepant (CAS: 1337918-83-8), VX-548 (CAS: 2649467-58-1), zatebradine, zotarolimus, deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0141] In certain embodiments, the therapeutic agent is a protein inhibitor or antagonist, including isotopomers thereof, such as deuterium-substituted derivatives.

[0142] Protein inhibitors or antagonists may target any suitable protein, including all variants, mutations, splice variants, indels and fusions of these listed target proteins. Examples include, but are not limited to, 5HT2c receptor, α 1A-AR, alpha-2 adrenergic receptor, alpha-synuclein, AAK1, ATP-binding cassette (ABC) transporters (such as MDR1 / 2 / 3 / 4 / 5 and ABCG2), ABL, cholesterol acyltransferase (ACAT), ACE protein, ACK, ACTR2, ACTR2B, ADCK1 / 2 / 3 / 4 / 5, protein kinase B (AKT), alphaK1 / 2 / 3, anaplastic lymphoma kinase (ALK), ALIX, amnionless, AMPA receptor (AMPAR), AMP-related protein kinase (AMPK), amyloid beta, angiotensin II receptor, ANKRD3, Apaf-1, apolipoprotein E receptor (ApoER), amyloid precursor protein (APP), androgen receptor (AR), ARAF, ARG, AR-V7, ileal apical sodium / bile acid cotransporter (ASBT), V-type proton ATPase 6 (ATP6V), ATP6V1 H, avidin, amino acid transporter, alanine serine cysteine ​​transporter (ASCT), ASGPR, ASK1 / 2, ataxin-1, ataxia telangiectasia mutated protein (ATM), ATM and Rad3-related protein (ATR), Aurora kinase, AXL, beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), beta2-adrenergic receptor, BAD, BARK1 / 2, Bax, BCKDK, B cell leukemia / lymphoma (BCL) family proteins (such as BCL2, BCL-XL and MCL-1), BCR-ABL, bromodomain and extraterminal domain family proteins (BET) (such as BRD2 / 3 / 4 / T), beta-catenin, BIKE / BMP2K, BLK, BMPR1A, BMPR1B, BMPR2, BMX / ETX, CDO brother of CDO,Boc), BRD9, BMI1, BRAF, BRAF V600E, brassinosteriod insensitive 1 (BRI1), BRK, BRM, BRSK1, BRSK2, Bruton's tyrosine kinase (BTK), BUB1, C3G, calreticulin, cell adhesion molecule (CAM) receptors, CAMK1α / β / γ / δ, CAMK2α / β / γ / δ, CAMK4, caMLCK, cannabinoid-1 receptor, coxsackievirus-adenovirus receptor (CAR), CBL-B, CRISPR-associated protein (Cas), CASK, caspase-3 (Caspa se-3), caspase-6, caspase-7, caspase-9, CBFβ, CBL, CBP, chemokine receptors (such as CC chemokine receptors (CCR) and CXC chemokine receptors (CXCR), such as CXCR2, CXCR4 and CXCR7), CCK4 / PTK7, CCR2, CCR9, CCRK, cluster of differentiation (CD) (such as CD4, CD9, CD13 / APN receptor, CD25, CD26 (DPP4), CD30, CD33, CD36, CD38, CD46, CD56, C D63, CD71, CD74, CD80, CD81, CD82, CD83, CD86, CD123, CD138, CD147, CD152, CD152(CTLA4), CD166, CD174, CD197, CD205, CD 227, CD228, CD269, CD276 and CD326), CDC7, CDC20, CDC25, CDC37, CDK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 / 16 / 17 / 18, CD KL1 / 2 / 3 / 4 / 5, CDON, CEACAM5, cholesteryl ester transfer protein (CETP), c-Fos, cystic fibrosis transmembrane conductance regulator (CFTR), cyclic GMP-AMP synthase (cGas), Chak1 / 2, CHK1 / 2, CI-M6PR, CK1α, CK1α2, CK1δ, CK1ε, CK1γ1 / 2 / 3, CK2α1 / 2, CLIK1, CLIK1L, Clip, CLK1 / 2 / 3 / 4, human collectin placenta 1 (CL-P1), CMYC, cone opsin (cone opsin)opsin), COT / TPL2, cell-penetrating peptide (CPP), connexin, coronavirus protease, CRABP, C-RAF, cereblon (CRBN), CREB, CRK, CRIK, CRIPTO, CSF2R, CSF1R / FMS, CSK, C-TAK1, CTK, pinocytosis receptor (cubilin), cyclin D, cyclin E, CytoC, DAPK1 / 2 / 3, DCAMK L1 / 2 / 3, DDR1 / 2, diacylglycerol acyltransferase (DGAT), DLK, DMPK1 / 2, DNA-PK, DP-1, DPP-4, DRAK1 / 2, deubiquitinating enzyme (DUB), DYRK1A / 1B / / 2 / 3 / 4, E2F, excitatory amino acid carrier 1 (EAAC1), E-cadherin, endothelin-converting enzyme (ECE), ED-B, EED, EF2K, eEF2K, EIF2A3, EIF4E, EIF4G, ELK, ENL, endothelin B receptors, epithelial cell adhesion molecule (EpCAM), endothelial protein C receptor (EPCR), ephrin receptor (EphR), estrogen receptor (ER), ER81, ErbB2, ErbB3, ETS transcription factor (ERG), ERK1 / 2 / 3 / 4 / 5 / 7 / 8, ERRa, EZH2, fatty acid binding protein (FABP), FADD, FAK, FAP, fatty acid transport protein (FATP), neonatal Fc receptor (FCRN), FER, FKBP, FLIP, folate receptor folate transporters (such as reduced folate carriers, FOLT1 / 2 / 3 and proton-coupled folate transporter (PCFT)), FOXM1, FOXO1 / 2 / 3 / 4, FRK, Frizzled4, fructose-1,6-bisphosphatase, FYN, gamma-aminobutyric acid type A receptor (GABAAR), GAK, Gap-1, growth arrest-specific 1 (Gas1), GCC, GCK, GCN2, GD2, ghrelin receptor (ghrelinR), glucose-dependent insulinotropic polypeptide (GIP), GloboH, glucagon receptor, glucagon-like peptide 1 (GLP-1), glutamate transporter (GLT), glucokinase, ionotropic AMPA glutamate receptor (GluR), glucose-6-phosphatase, glucose transporter (GLUT), glutathione transporter, glycogen phosphorylase, glycogen synthase kinase, glycoproteins (such as gp18, gp31 and gp60), G protein-coupled receptors (GPCRs) (such as GPR20, free fatty acid receptor 1 (GPR40), GPR119 and GPR120), GP NMB, GPRK4 / 5 / 6 / 7, GRB2, GSK-3, GSPT1, Haspin kinase, HCK, hepatitis C virus (HCV) NS3 / 4A, histone deacetylase (HDAC), HDL receptor (HDLR), HectH9, HH498, HIF1, HIPK1 / 2 / 3 / 4, HMGN1, HMG-CoA reductase, hnRNP, H-PGDS, HPGCR, hematopoietic progenitor cell kinase 1 (HPK1), HRAS, HRI, hRpn13 Pru , HSD-11β (11β-hydroxysteroid dehydrogenase), heat shock protein (HSP), huntingtin protein (HTT), HUNK, ICK, intermediate density lipoprotein receptor (IDLR), IDO-1, IFN-γR, IGF-1R, IGF-2R, IKK-α, IKK-β, IKK-γ, IKK-ε, Ikaros (IKZF1), Helios (IKZF2), Aiolos (I KZF3), inhibitor of apoptosis proteins (IAPs) (such as cIAP and XIAP), IKZF4, IL-4R, IL-10R, ILK, integrins (such as αVβ3, α4β1, and α5β1 integrins), insulin receptor (IR), insulin-like growth factor receptor, INSR, IRE1 / 2, IRR, ITKIMP, integrin receptor (IR), IRA2, IRAK1 / 2 / 3 / 4, IRE1, Janus tyrosine kinase (JAK), c-Jun N-terminal kinase (JNK), potassium-chloride cotransporter 2 (KCC2), KDM5C, KHS1 / 2, inwardly rectifier potassium channel (Kir2.3), KIS, KIT, Kelch domain-containing protein 2 (KLHDC2), KLF4, KRAS, KRAS G12C, KSR1 / 2, lamin, lysosomal-associated membrane protein (Lamp), LAMTOR2, LANA, lactoferrin receptor, L-amino acid transporter (LAT), LATS1 / 2, LCK, low-density lipoprotein receptor (LDLR), LEF, LIMK1 / 2, LKB1, LMR1 / AATK, LMR2 / 3, LOK, low-density lipoprotein receptor-related protein (LRP), LRRK1 / 2, LTK, LXR-β, LYN, LZK, LZTFL1, m5C, m6A, MAK, MALT, MAP3K4 / 8, MAP4K3, MAP4K5, MAPK, MAPKlb, MAPKAPK2 / 3 / 5, MARK1 / 2 / 3 / 4, MAST1 / 2 / 3 / 4 / L, MC4 receptor, MCAK, MCH-1 receptor, monocarboxylate transporter (MCT), mouse double minute 2 homolog (MDM2), MDMx, megalin, mitogen-activated protein kinase (MEK), MEKK1 / 2 / 3, MEL, MELK, MER, MERTK, mesothelin, MET, Mfsd2a, metabotropic glutamate receptor (mGlu1), major histocompatibility complex class I protein (MHC-I), MHC-II, MIF, MINCLE, MINK1, MISR2, MITF, MKK1 / 2 / 3 / 4 / 6 / 7, MKP3, MLK1 / 2 / 3 / 4, MLKL, MLN1 / 3, MNK1 / 2, MOK, MOS, MPK, MPSK1, MRCKα / β, multidrug resistance protein (MRP), MSK1 / 2, MSSK1, MST1 / 2 / 3 / 4, mammalian target of rapamycin (mTOR), MUCL, MUSK, Myc protein, MYO3A / 3B, MYT1, NAMPT, N-cadherin, K + Dependent Na + / Ca 2+exchanger 2 (NCKX2), NDC80, NDR1 / 2, NEK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11, NF-κB, Nicastrin, nicotinic acetylcholine receptor, NIK, NLK, NOTCH receptor, Niemann-Pick C1-like 1 1,NPC1L1), N-methyl D-aspartate receptor (NR), NRAS, NRBP1 / 2, NRF2, NSD2, NSD3, nucleoside transporter (NT), sodium / taurocholate co-transporting peptide (NTCP), NuaK1 / 2, NUAK1, organic anion transporter (OAT), organic anion transporting polypeptide (OATP; OATP1B1, OATP2B1, OATP4C1, etc.), obscurin, organic cation transporter (OCT), OSR1, organic solute transporter (OST), otoferlin, P2X purinergic receptor 4 (P2x4R), purinergic receptor P2Y12 (P2Y12), p16INK4A, p18INK4, p19INK4D, p21, p27 kip1, p38α, p38β, p38γ, p38δ, p53, p65 / RELA, p70S6K, p70S6Kb, p90, p300 protein, PAK1 / 2 / 4 / 5 / 6, pan-coronavirus antiviral, protease-activated receptor (PAR), PARP, PASK, PAX, PBRM1, PCSK9, PD-1, phosphodiesterase (PDE), PDE4, PDEδ, PDGFRα / β, pdhk1 / 2 / 3 / 4, PDK1, PD-L1, PEA-15, PEK, peptide transporter (PEPT), P-glycoprotein (P-gp), PHKy1 / 2, phosphoenolpyruvate carboxykinase, phosphoinositide 3-kinase (PI3K), PIM1 / 2 / 3, PINK1, PITSLR E, PKA, PKACα / β / γ, PKBα / β, PKC, PKD1 / 2 / 3, PKG1 / 2, PKN1 / 2 / 3, PKR, PLC, PLDL, PLK1 / 2 / 3 / 4, PMEL17, PRAK, peroxisome proliferator-activated receptor (PPAR), PRC2, PRK2, PRKX, PRKY, PRMT5, protein phosphatase, protein tyrosine phosphatase (PTP), PRP4, PRPK, PSKH1 / 2, prostate-specific membrane antigen (PSMA), receptor patched 1 (PTCH1), PTEN, protein tyrosine kinase (PTK), PVRL4, pyruvate dehydrogenase kinase, PYK2, QIK, QSK, R1P1, RACK-1, Rac protein, RAF protein, RAP1, Raptor, RAR, Ras protein family (RAS), Rb, retinol binding protein (RBP), RET, riboflavin transporter protein (RFVT), RFX1, RHAU, RHODK, RIOK1 / 2, receptor interacting protein kinase (RIPK), rhodopsin, ribonuclease K (RNASEK), RNAseL, RING finger protein (RNF), ROCK1 / 2, renal outer medullary potassium channel (ROMK), RON, ROR1 / 2, ROS, RPN11, RPN13, ribosomal s6 kinase, receptor serine / threonine kinase (RSK) (such as transforming growth factor beta (TGF-β) receptor and receptor serine / threonine kinase (RSK)) (such as Aristidis Moustakas et al., receptor serine / threonine kinase) Serine / ThreonineKinases) (ISBN: 978-3-540-44244-8), receptor tyrosine kinases (RTKs) (such as fibroblast growth factor receptor (FGFR), FLT1 / 2 / 3 / 4, FGFR1 / 2 / 3 / 4, RET, tropomyosin-related kinase (TRK), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER), vascular endothelial growth factor receptor (VEGFR), TYRO3 and as listed in the publication Mark A. Lemmon and Joseph Schlessinger, Cell, 141(7)1117-1134, 2010), RUNX1 / 2 / 3, RYK, spindle assembly checkpoint (SAC), Sanpodo / Notch, SAP, SARS-CoV-2, SBK, solute carrier (SC), SCF, SCYL1 / 2 / 3, SF3B1, serine / threonine protein kinase (SGK), SGLT, SHC, SHP2, SIK2 / 3, SIRT2, SIX1, skMLCK, S phase kinase-associated protein 1 (SKP1), S phase kinase-associated protein 2 (SKP2), solute carriers (SLC, such as SCL19A1) transporters (such as hMATE1 and as in the publication Enrico Girardi et al., Nature Chemical Biology, 16, 469-478, 2020), SLK, SLOB, Smac, Smad proteins, SMARCA, sodium-coupled monocarboxylate transporter (SMCT), SMG1, smMLCK, sodium-dependent multivitamin transporter (SMVT), synaptosome-associated protein (SNAP), SNRK, sortilin-related CNS expressed 1a (SorCS1a), SorCS1c, son of sevenless (SOS), SOS1, SOX2, secreted protein acidic and rich in cysteine ​​(SPARC), SPEG, speckled BTB / POZ protein (SPOP), SPRED, SPRY, SPv-NK1R, scavenger receptors (SR) (such as CD36, LAMP1 and LAMP2), Src proteins, SRF, SRM, SRPK1 / 2, SSTK, StaO, signal transducer and activator of transcriptiontranscription protein, STAT), STING, serine / threonine kinase (STK), STLK3 / 5 / 6, syntaxin (STX), SuRTK106, SUV39HI, SUZ12, sodium-vitamin C cotransporter (SVCT), synaptotagmin (SYT), triiodothyronine (T3), TAB, TAK1, TAL, TAO1 / 2 / 3, tau protein, TBCK, TBK1, T cell factor / lymphoid enhancer binding factor (TCF / LEF family), TEC, TESK1 / 2, TFAM, transcription factor EB (TFEB), transferrin receptor (TfR), TGF-β, trans-Golgi network (trans-Golgi network) (TGN38), thiamine transporter (THTR), TIE1, TIE2 / TEK, TIF1a / β / γ, titin / TTN, TLK1 / 2, toll-like receptors (TLRs) (such as TLR4), TNK1, TOPK, TPL2 / COT, TPN1p, TRAD, TRAF proteins, Trb1 / 2 / 3, tripartite motif family proteins (TRIM), Trio, TRRAP, TSC2, TSG101, TSSK1 / 2 / 3 / 4, TTBK1 / 2, TTK, TXK, TYK2, tyrosinase, tubulin, UBF, UCHL5, ULK1 / 2 / 3 / 4, ubiquitin-specific peptidase (USP) (such as USP7, USP P11 and USP14), VACCAMKL protein, vesicle-associated membrane protein (VAMP), vitamin D receptor (VDR), vesicular GABA transporter (VGAT), vesicular glutamate transporter (VGLUT), very low-density lipoprotein receptor (VLDLR), urate transporter 1 (URAT1), VRK1 / 2 / 3, WAVE-2, WDR5, WEE-1, WEE-1B, WNK1 / 2 / 3 / 4, Wnt, XBP1, YANK1 / 2 / 3, YBX1, YES, YSK1, ZAK, ZAP70, ZC1 / 2 / 3 / 4, ZFP91 and zinc ring finger protein (ZRNF), including all variants, mutations, splice variants, indels and fusions of these target proteins listed.

[0143] In certain embodiments, the protein inhibitor or antagonist is a molecule targeting a B-cell lymphoma 2 (BCL-2) family protein. In certain embodiments, the protein inhibitor or antagonist targeting a BCL-2 family protein is selected from ABT737 (CAS: 852808-04-9), ABT263 (CAS: 923564-51-6), ABT199 (CAS: 1257044-40-8), GX15-070 (CAS: 803712-79-0), UBX1325 (lisatoc), APG-1252 (pesitoq), APG-2575 (lisatoc) or -(-)-gossypol (CAS: 90141-22-3), any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0144] In certain embodiments, the protein inhibitor or antagonist is a molecule targeting proprotein convertase subtilisin / kexin type 9 (PCSK9). In certain embodiments, the protein inhibitor or antagonist targeting PCSK9 protein is selected from CVI-LM001, PF-06815345, MK-0616 or enlicitide chloride (CAS: 2407527-16-4), NN6434, 13PCSK9i and any molecule listed in the publication Shakir Ahamad et al., Journal of Medicinal Chemistry, 65 (23), 15513-15539, 2022, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0145] In certain embodiments, the protein inhibitor or antagonist is a molecule that targets a protein phosphatase. In certain embodiments, the protein inhibitor or antagonist targeting protein phosphatase is selected from TNO155 (CAS: 1801765-04-7), RMC-4630, RMC-4550 (CAS: 2172651-73-7), IFB-088 (CAS: 951441-04-6), SHP1 inhibitor (CAS: 56932-43-5), SHP009 (CAS: 1801747-42-1), DPM-1001 (1471172-27-6), AKB-9778 (CAS: 1008510-37-9), LB-100 (CAS: 1026680-07-8), any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0146] In certain embodiments, the protein inhibitor or antagonist is a molecule that targets histone deacetylase (HDAC). In certain embodiments, the HDAC inhibitor or antagonist is selected from but not limited to vorinostat, romidepsin, belinostat (PXD101), panobinostat (LBH589), valproic acid (valproic acid), valproic acid, ... acid), entinostat (MS275), butyric acid, trichostatin A, givinostat (ITF2357), citarinostat (ACY-241), mocetinostat (MGCD0103), pracinostat (SB939), resminostat, RGFP966, CUDC-101, abexinostat (PCI-24781), nocetinostat, phenylbutyrate, tacedinaline, tubastatin, tubastatin A A), R306464, SE-7552, MPT0B451, dacinostat (LAQ824), HDAC10-IN-1, HDAC10-IN-2, AR-42, GSK3117391, MC1568, quisinostat (JNJ-26481585), PCI-34051, droxinostat, RGFP966, ricolinostat (ACY-1215), acetyldinaline (CI994), fimepinostat (CUDC-907), M344, RG2833 (RGFP109), scriptaid, TMP269, TMP195, santacruzamate A A)(CAY10683), SKLB-23bb, ACY-775, BRD73954, CXD101, suberohydroxamic acid, BRD3308, HPOB, LMK-235, nexturastat A, BML-210(CAY10433), KA2507, TC-H106, Tucidinostat (Chidamide), SIS17, WT161, CAY10603, ACY-738, tinostamustine (EDO-S101), domatinostat (4SC-202), BG45, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0147] In certain embodiments, the protein inhibitor or antagonist is a molecule that targets mammalian target of rapamycin (mTOR) and / or FK506 binding protein (FKBP). In certain embodiments, the mTOR and / or FKBP (or 4EBO1) inhibitor or antagonist is rapamycin, Rapalink-1 (CAS: 1887095-82-0) or RMC-5552 (CAS: 2382768-62-7), any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0148] In certain embodiments, the protein inhibitor or antagonist is a molecule targeting a protein methyltransferase, such as arginine methyltransferase (PRMT). In certain embodiments, the PRMT inhibitor or antagonist is selected from, but is not limited to, S-adenosylmethionine (SAM), S-adenosylhomocysteine ​​(SAH), AMI-1, sinefungin, homosinefungin, GSK3326595, JNJ-63619178, GSK3368715, EML108, EPZ004777, and EML981, any deuterium-substituted derivatives, analogs, and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0149] In certain embodiments, the protein inhibitor or antagonist is a molecule that targets signal transducer and activator of transcription (STAT).

[0150] In certain embodiments, the therapeutic agent is a protein activator or agonist and modulator, including their isotopomers, such as deuterated derivatives. Protein activators or agonists can target any suitable protein. Examples include, but are not limited to, adenosine A1 receptor (A1R), A 2A Receptor, A 2Breceptors, A3 receptors, AMPK, cGAS, chemokine receptors, FXR, GCK, glucagon-like peptide 1 (GLP-1), G protein-coupled receptors (GPCRs) (such as GPR20, GPR20, free fatty acid receptor 1 (GPR40), GPR119 and GPR120), integrins, peroxisome proliferator-activated receptors (PPARs), RING finger proteins (RNFs), stimulator of interferon genes (STING), Toll-like receptors (TLRs) (such as TLR4), zinc RING finger proteins (ZRNFs), including all variants, mutations, splice variants, indels and fusions of these target proteins listed.

[0151] In some embodiments, the activator or agonist is a molecule that targets the adenosine A1 receptor (A1R). In certain embodiments, the A1R activator or agonist is selected from, but not limited to, CPA (CAS: 41552-82-3), BnOCPA, LUF6258, and VCP746 (CAS: 1582751-84-5), any deuterium-substituted derivatives, analogs, and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0152] In certain embodiments, the activator or agonist is a molecule that targets glucagon-like peptide 1 (GLP-1). In certain embodiments, the GLP1 activator or agonist is selected from, but is not limited to, Lixisenatide / AVE0010 / ZP10 / Lyxumia, Exenatide / Exendin-4 / Byetta / Bydureon / ITCA650 / AC-2993, Liraglutide / Victoza, Tirzepatide, Semaglutide, Taspoglutide, Syncria / Albiglutide, Dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054, Efpeglenatide / HM-11260C, Efpeglenatide CM-3, GLP- 1Eligen, ORMD-0901, NN-9924, NN-9926, NN-9927, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD- 1. GSK-2374697, DA-3091, MAR-701, MAR709, ZP-2929, ZP-3022, ZP-DI-70, TT-401, MK-8521, MED103 82, BHM-034, HM12525A, MOD-6030, CAM-2036, DA-15864, ARI-2651, ARI-2255, LY3298176, NN1177, exenatide-XTEN and glucagon-XTEN, NN9030, and any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0153] In certain embodiments, activators or agonists and modulators are molecules that target integrins. In certain embodiments, integrin activators or agonists and modulators are selected from, but are not limited to, SAR-1118, BMS-587101, 1,2,3,4-tetrahydroquinoline-6-carboxylic acid, HC-0303, Compactin, AJM-300, HMR-1031, Firalast, Tirofiban, Eptifibatide, MK-0429, ATN-161, JSM-6427, and any deuterium-substituted derivatives, analogs, and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0154] In certain embodiments, the activator or agonist is a molecule that targets peroxisome proliferator-activated receptors (PPARs). In certain embodiments, the PPAR activator or agonist is selected from but not limited to GW0742, L-165041, MA-0211, KD-3010, CER-002, SAR351034, Oxeglitazar, LY518674, ZYH7, K111, Macuneos, Efatutazone, CHS-131, OMS-405, GED 0507-34-Levo, T2D 959, Lanifibranor, Gemfibrozil, Rosiglitazone, Ciprofibrate, Pioglitazone, Bezafibrate, Lobeglitazone, Fenofibrate, Saroglitazar, Pemafibrate, any deuterium-substituted derivative, analog and chelate, or pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and a pharmaceutically acceptable carrier.

[0155] In certain embodiments, the activator or agonist is a molecule that targets the stimulator of interferon genes (STING) protein. In certain embodiments, the STING activator or agonist is selected from, but not limited to, c(di-GMP), 3',3'-cGAMP, 2',3'-cGAMP, ML-RR-S2-cGAMP, ADU-S100, ML-RR-S2-CDG, DMXAA, aminobenzimidazole, ExoSTING, MV-626, SB11285, STACT-TREX1, SYN-STING (SYNB1 891), E7766, GSK3745417, MK-1454, MK-2118, BMS-986301, SB-11285, IMSA-101, BI-1387446, TAK676, SNX281, HG-381, DN-015089, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0156] In certain embodiments, the therapeutic agent is a pyrophosphate or bisphosphonate-containing agent that can bind to bone mineral and be taken up by bone cells via membrane receptors such as SCL37A3. In certain embodiments, the pyrophosphate or bisphosphonate-containing agent is selected from, but is not limited to, pamidronate, risedronic acid, alendronic acid, zoledronic acid, ibandronic acid, minodronic acid, publications Jaeok Park et al., Frontiers in Chemistry, 8, 612728, 2021 and Zhou Yu et al., eLife, e36620, 2018, any deuterated derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0157] In certain embodiments, the therapeutic agent is a molecular glue or degrader, including their isotopomers, such as deuterated derivatives. A molecular glue can be any compound that can stabilize the interaction between two or more proteins. A degrader can be any compound that can bind to a protein of interest (POI) and induce degradation of the POI by directly modulating the POI (such as modifying the surface topology of the POI).

[0158] In some embodiments, the therapeutic agent is a multivalent endocytosis agent. The multivalent endocytosis agent herein is an agent or probe. Via cleavable or non-cleavable chemical bonds or linker units In any equivalent and order with chemical arms The multivalent endocytosis agent can bind to the membrane component that mediates endocytosis and be absorbed via endocytosis. The binding affinity K of the multivalent compound for the membrane component that mediates endocytosis is D Can be less than 20.0mM.

[0159] In certain embodiments, multivalent endocytic agents include, but are not limited to, structures as represented by formula (I):

[0160]

[0161] wherein m, n and p represent an integer from 0 to 100. Each of m, n and p may independently be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0162] In certain embodiments, the multivalent endocytosis agent itself can exert biological functions in bacteria, viruses, fungi, protozoa, vectors, cells, tissues and animals. In certain embodiments, the chemical bonds or linker units in the multivalent endocytosis agent It can be cleaved by enzymes (or proteins) in bacteria, viruses, fungi, protozoa, vectors, cells, tissues, and animals, and a portion of the multivalent endocytic agent is released to exert any biological function. Components in the internalized membrane and extracellular materials, such as proteins, carbohydrates, lipids, pathogens, particles, viruses, bacteria, fungi, protozoa, vectors, cell debris, and other cells, can also be degraded by lysosomes.

[0163] The agent and the chemical arm are connected by chemical bonds or linker units Covalent conjugation. In certain embodiments, the chemical bond or linker unit Can be connected to drugs or probes and chemical arm at any location.

[0164] In certain embodiments, the conjugation site between the agent or probe and the chemical arm can be selected to allow, improve, or enhance the intended function of the agent while allowing, improving, or enhancing the binding affinity to the membrane component that mediates endocytosis to enhance endocytic efficacy and / or efficiency. For example, conjugation between the agent or probe and the chemical arm produces a multivalent endocytic agent.

[0165] The conjugation site between the agent or probe and the chemical arm can be selected to impair, reduce or eliminate the intended function of the agent.

[0166] In certain embodiments, the chemical bond or linker unit It can be a polyvalent polyethylene glycol, alkoxy, alkyl, alkylene, alkyne, cycloalkyl, cycloalkylene, cycloalkyne, heterocyclyl, aryl, heteroaryl, which can be terminated (at one or both ends) with at least one of the following: -H, -D, -O(R), =O, -S(R), =S, =NR, =N(R), -N(R)(R'), -Se(R), =Se, -F, -Cl, -Br, -I, -F, - 18F, -C(R)(R')(R”), =C(R)(R’), ≡C(R), -Si(R)(R’)(R”), =Si(R)(R’), ≡Si(R), -OS(O)2R, -OS(O)R, -S(O)R, -S(O)2R , -P(O)(R)(R'), -P(R)(R'), -P(O)(OH)O-, -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(S)O-, - OC(S)-, -OC(S)O-, -C(NOR)-, -C(O)N(R')-, -C(O)N(R)C(O)-, -C(O)N(R)C(O)N(R')-, -N(R)C(O)-, -N(R')C(O)N(R )-, -N(R)C(O)O-, -OC(O)N(R)-, -C(NR')-, -N(R)C(NR')-, -C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(O)2-, -OS(O)-, - S(O)O-, -S(O)-, -OS(O)2--S(O)20-, -N(R)S(O)2-, -S(O)2N(R)-, -N(R')S(O)-, -S(O)N(R)-, -N(R)S(O)2N(R')-, - N(R')S(O)N(R')-, -SS-, -O-Si(R)(R')-O-,)-, -C(=N)N(R)(R')-, -C(R)=C(R')-, -C≡C-, C3-12 cycloalkane, C3-12 cycloalkene, C3-12 cycloalkyne, a 3- to 12-membered heterocycle, a 5- to 12-membered aryl, a 5- to 12-membered heteroaryl, any deuterium-substituted derivative, or any combination thereof, wherein R, R', or R" is H, D, a 1-100 polyethylene glycol, a C1-C100 alkoxy, a C1-C100 alkyl, a C2-C100 alkylene, a C2-C100 alkyne, a C3-C100 cycloalkyl, a C3-C100 cycloalkylene, a C3-C100 cycloalkyne, a C3-C100 heterocyclyl, a C6-C100 aryl, or a C1-C100 heteroaryl, wherein one or two end-capping groups may be the same or different.

[0167] In certain embodiments, the chemical bond or linker unit Can be a multivalent chain unit It contains one or more than one chemical bond or linker unit Cores connected in any quantity and order wherein m and n represent integers from 0 to 100.

[0168] In certain embodiments, the core Including but not limited to atoms H, C, Si, N, P, B, O, S, Se, 1-100 polyethylene glycol, C1-C100 alkoxy, C1-C100 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkylene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl or C1-C100 heteroaryl, any deuterium substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0169] In certain embodiments, the chemical bond or linker unit It can be a divalent or trivalent polyethylene glycol, alkoxy, alkyl, alkylene, alkyne, cycloalkyl, cycloalkylene, cycloalkyne, heterocyclyl, aryl, heteroaryl, which can be terminated (at one or both ends) with at least one of the following: -H, -D, -O(R), =O, -S(R), =S, =NR, =N(R), -N(R)(R'), -Se(R), =Se, -F, -Cl, -Br, -I, -F, - 18- P(O)(R)(R'), -P(R)(R'), -P(O)(OH)O-, -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(S)O-, -OC(S) -, -OC(S)O-, -C(NOR)-, -C(O)N(R')-, -C(O)N(R)C(O)-, -C(O)N(R)C(O)N(R')-, -N(R)C(O)-, -N(R')C(O)N(R)-, -N(R )C(O)O-, -OC(O)N(R)-, -C(NR')-, -N(R)C(NR')-, -C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(O)2-, -OS(O)-, -S(O)O-, -S( O)-, -OS(O)2-, -S(O)2O-, -N(R)S(O)2-, -S(O)2N(R)-, -N(R')S(O)-, -S(O)N(R)-, -N(R)S(O)2N(R')-, -N(R')S(O)N(R')-, -SS-, -O-Si(R)(R')-O-,)-, -C(=N)N(R)(R')-, -C(R)=C(R')-, -C≡C-, C3-12 cycloalkane, C3-12 cycloalkene, C3-12 cycloalkyne, 3 to 12 membered heterocycle, 5 to 12 In some embodiments, m or n is an integer from 0 to 50.

[0170] In certain embodiments, the linker is a cleavable bond. Cleavable bonds include, but are not limited to, phosphates, amides, esters, dialkyl or diaryl dialkoxysilanes, cyanoethyl, sulfones, ethylene, glycolyl disuccinates, cyclic acetals, 2-N-acylnitrobenzenesulfonamides, α-thiophenyl esters, unsaturated vinyl sulfides, sulfonamides, malondialdehyde (MDA)-indole derivatives, levulinic acid esters, hydrazones, oximes, imides, acylhydrazones, alkylthioesters, thioesters, disulfide bridges, azo compounds, 2-nitrobenzyl derivatives, benzoylmethyl esters, 8-quinolylbenzenesulfonates, coumarins, bis-arylhydrazones, bimane bi-thiopropionic acid derivatives, and the like. acid), p-methoxybenzyl derivatives, tert-butyl carbamate analogs, orthoesters, acetals, aconityl, silyl ethers, β-thiopropionates, phosphoramidates, disulfides, vinyl ethers, polyketals, allyl esters, picolinates, vicinal diols, and selenium compounds.

[0171] In certain embodiments, the agent or probe It is a part of a drug or therapeutic agent or diagnostic agent or chemical probe, and is any deuterium-substituted derivative, analog and chelate, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and a pharmaceutically acceptable carrier.

[0172] In certain embodiments, the chemical arm An atom or group of atoms, a compound or a portion of a compound that can bind to a membrane component or extracellular material, including a membrane protein, an extracellular protein, a carbohydrate, a lipid, a pathogen, a particle, a virus, a bacterium, a fungus, a protozoan, a vector, a cell fragment, and another cell. Thus, a multivalent endocytic agent can be used to internalize a cell membrane to form an endosome, and then transport the multivalent endocytic agent itself, the membrane protein, the extracellular protein, a carbohydrate, a lipid, a pathogen, a particle, a bacterium, a virus, a fungus, a protozoan, a vector, a cell fragment, and another cell into the cell or into a lysosome of the cell.

[0173] In certain embodiments, the chemical arm It is a binding moiety that can bind to a membrane component that mediates endocytosis, wherein the chemical arm is selected from but not limited to an atom, a chemical bond (including a reversible or irreversible covalent bond), a chemical moiety that is charged or chargeable (at a specific pH value), a hydrophilic moiety, a lipophilic moiety, a chemical moiety containing a reversible or irreversible covalent bond, an agent, a portion of an agent, a membrane-binding chemical fragment (MBCF), substituted mono- or dicarboxylic acid derivatives (SMDA), lipids and derivatives (LA), substituted phosphate derivatives, glycerides and derivatives (GA), phospholipids and derivatives (PPA), steroids, vitamins and derivatives (VtA), amino acids and peptides and derivatives (AAP), monosaccharides, sugars and derivatives (SCA), nucleobases and nucleosides and nucleotide derivatives (NNNA), reported membrane protein binders and derivatives (RMPB), any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0174] In certain embodiments of the membrane-binding chemical fragment, substituted mono- or dicarboxylic acid derivatives (SMDA) include, but are not limited to, saturated or unsaturated alkyl or heteroalkyl chains, which also include chains having 1 to 50 terminal carboxylic acid moieties. Without limitation, the alkyl or heteroalkyl groups having a terminal carboxylic acid moiety are represented by propionic acid, butanoic acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecenoic acid, lauric acid, tridecanoic acid, myristic acid, palmitic acid, hexadecanoic acid, heptadecanoic acid, stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, tetradecenoic acid, pentadecanoic acid, palmitoleic acid, oleic acid, eicosenoic acid, docosenoic acid, tetracosanoic acid, eicosapentaenoic acid, docosatrienoic acid, docosahexaenoic acid, octadecadienoic acid, octadecatrienoic acid, eicosadienoic acid, eicosatrienoic acid, eicosatetraenoic acid, mevalonate acid, carotenoic acid, retinoic acid, dihydroretinoic acid, fenofibric ... acid), any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0175] In certain embodiments of the membrane-bound chemical segment, the terminal carboxylic acid moiety can be replaced with, but is not limited to, hydroxamic acid, hydroxamate, hydroxamic acid amide, carbonic acid, carbonate, carbonate amide, sulfonic acid, sulfonic acid ester, sulfonic acid amide, sulfurous acid, sulfurous acid ester, sulfurous acid amide, nitric acid, nitrate, nitric acid amide, nitrous acid, nitrite, nitrite, nitrite amide, boric acid, boric acid ester, boric acid amide, phosphoric acid, phosphate ester, phosphoric acid amide, phosphorous acid, phosphite, phosphite amide, phosphinic acid, phosphinic acid ester, phosphinic acid amide, pyrophosphoric acid, pyrophosphate, pyrophosphate amide, alcohol, aldehyde, amine, any deuterium-substituted derivative, analog and chelate, or a pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and a pharmaceutically acceptable carrier.

[0176] In certain embodiments of membrane-binding chemical fragments, lipids and derivatives (LA) include but are not limited to lipids optionally substituted with 0-6 R 1 substituted saturated or unsaturated C4-C 100 Alkyl chain, saturated or unsaturated C4-C 100 Heteroalkyl chains, glycerides, phospholipids, ceramides, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0177] In certain embodiments of the membrane-binding chemical fragment, saturated or unsaturated alkyl or heteroalkyl chains include, but are not limited to, phytoene, phytofluene, neurosporene, lycopene, didehydrolycopene, apolycopene, aponeurosporene, diaponeurosporene, cryptoxanthin, lutein, zeaxanthin, phytoene, carotenoid aldehydes, retinal, squalene, squalane, squalene 2,3-oxide, squalene 2,3:22,23-dioxide, polyp odatetraene), isodammara-20(21),24-diene, isodammara-12,24-diene, dammara-13(17),24-diene, euphorbia-7,24-diene, damma-20(21),24-diene, oxidosqualene, farnesol, farnesyl acetate, 11-hydroxy-10,11-dihydrofarnesyl acetate, 10-bromo-11-hydroxy-10,11-dihydrofarnesol, 10,11-epoxyfarnesyl acetate, 10,11-epoxyfarnesol, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0178] In certain embodiments of the membrane-binding chemical fragment, glycerides and their derivatives (GA) include, but are not limited to, monoerucin, monolaurin, monomyristin, monopalmitin, monostearin, 1,3-dioleoyl-2-palmitoyl-glycerol, 1,3-dipalmitolein, 1,2-diolein, 1,3-diarachidonin, 1,3-dipalmitolein, 1,2-dipalmitolein, 1,3-distearin, tripalmitolein, trielaidin, tripetroselaidin, trilinolein, trimyristin, tripalmitolein, Tristearin, 1,3-dipalmitelaidin, 2-acetyl-1,3-dicaffeoylglycerol, 2-acetyl-1-caffeoyl-3-coumaroylglycerol, 2-acetyl-1-feruloyl-3-caffeoylglycerol, 2-acetyl-1-feruloyl-3-coumaroylglycerol, 2-acetyl-1,3-dicaffeoylglycerol, 2-acetyl-1-caffeoyl-3-cinnamoylglycerol, 2-acetyl -1,3-dicoumaroylglycerol, 2-acetyl-1-coumaroyl-3-feruloylglycerol, acetylcoumaroylglycerol, coumaroylglycerol, 1,3-dicoumaroylglycerol, 1-coumaroyl-3-caffeoylglycerol, caffeoylglycerol, tricoumaroylglycerol, coumaroylferuloylglycerol, dicaffeoylcoumaroylglycerol, dicaffeoylferuloylglycerol, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0179] In certain embodiments of the membrane-binding chemical fragment, phospholipids and their derivatives (PPA) include but are not limited to phosphatidic acid, cardiolipin (CL), lysobisphosphatidic acid (LBPA), lysophosphatidic acid (LPA), phosphatidylcholine (PC), phosphatidylserine (PtSer), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidyserine (PS), phosphatidylinositol (PI), phosphatidylinositol, sphingomyelin, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0180] In certain embodiments of the membrane-binding chemical fragment, steroids include, but are not limited to, cholesterol, ergosterol, lithocholic acid, 7-dehydrocholesterol, 22,23-dihydroergosterol, 7-dehydrositosterol, 7-dehydrostigmasterol, 7-dehydrocamperterol, pregnenolone, 17α-hydroxypregnenolone, 16α-hydroxypregnenolone, 20α-dihydropregnenolone, dehydroepiandrosterone (DHEA), 7α-hydroxy-DHEA, 7-oxo-DHEA, 7β-hydroxy-DHEA, 5-androstene-3β,17β-diol, 5-androstene- 3β,7α,17β-triol, 5-androstene-3β,7β,17β-triol, 5-androstene-3β,16α,17β-triol, progesterone, 17α-hydroxyprogesterone, 17α,20α-dihydroxy-4-pregnen-3-one, 16α-hydroxyprogesterone, 20α-dihydroprogesterone, androstenedione, testosterone, 16α-hydroxytestosterone, 5α-dihydrotestosterone, estrone, estradiol, estriol, 5α-dihydroprogesterone, allopregnanolone, isopregnanolone, 5β-dihydroprogesterone, pregnanolone, epipregnanolone, 5α,20α-tetrahydroprogesterone, 5α-pregnane-3α,20α- diol, 5α-pregnane-3β,20α-diol, 5β,20α-tetrahydroprogesterone, 5β-pregnane-3α,20α-diol, 5β-pregnane-3β,20α-diol, 17α-hydroxyallopregnanolone, 17α-hydroxypregnanolone, 5α-pregnane-3α,17α,20α-triol, 5α-pregnane-3β,17α,20α-triol, 5β-pregnane-3α,17α,20α-triol, 5α-androstane-3,17-dione, androstenone, epiandrostenone, etiocholanolone, 5α-androstane-3α,17β-diol, 5α-androstane-3β,17β-diol, cortisol, cortisone, corticosterone, 21- Deoxycortisol, 11-deoxycorticosterone, 3α,5α-tetrahydrocorticosterone, 3α,5β-tetrahydrocorticosterone, 11β-hydroxyandrostenedione, tetrahymanol, 11β-hydroxyandrostenone, 11β-hydroxyepiandrosterone, 11β-hydroxybencholanolone, hopene, hopeanol, hopan-22(29)-ene, hopan-22-ol, diplopterol, tetrahymanol, bacteriohopaneteirol, aminobacteriohopanetriol, lanosterol, 24,25-Oxylanosterol, 24,25-Epoxycholesterol, Pregnenolone Sulfate, 17α-Hydroxypregnenolone Sulfate, 20α-Dihydropregnenolone Sulfate, DHEA Sulfate, Androstenediol Sulfate, 5-Androsten-3β,16α,17β-Triol Sulfate, Conjugated 17α,20α-Dihydroxy-4-Pregnen-3-one, Conjugated 20α-Dihydroprogesterone, Conjugated Testosterone, Conjugated Epitestosterone, Estrone Sulfate, Estradiol Sulfate, Sulfate Estriol sulfate, allopregnanolone sulfate, allopregnanolone sulfate, conjugated pregnanolone, conjugated epipregnanolone, conjugated 5α,20α-tetrahydroprogesterone, conjugated 5α-pregnane-3α,20α-diol, conjugated 5α-pregnane-3β,20α-diol, conjugated 5β,20α-tetrahydroprogesterone, conjugated 5β-pregnane-3α,20α-diol, conjugated 5β-pregnane-3β,20α-diol, 17α-hydroxy Allopregnanolone, conjugated 17α-hydroxypregnanolone, 5α-pregnane-3α,17α,20α-triol, 5α-pregnane-3β,17α,20α-triol, 5β-pregnane-3α,17α,20α-triol, androsterone sulfate, epiandrosterone sulfate, bencholanolone sulfate, epibencholanolone sulfate, conjugated 5α-androstane-3α,17β-diol, conjugated 5α-androstane-3β,17β-diol, conjugated 5β- Androstane-3α,17β-diol, conjugated 5β-androstane-3β,17β-diol, conjugated 3α,5α-tetrahydrocorticosterone, conjugated 3α,5β-tetrahydrocorticosterone, 11β-hydroxyandrosterone sulfate, 11β-hydroxyepiandrosterone sulfate, 11β-hydroxybencholanolone sulfate, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0181] In certain embodiments of the membrane-binding chemical fragment, vitamins and derivatives (VtA) include, but are not limited to, coenzyme Q10, vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenate), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B8 (inositol), vitamin B9 (folic acid), vitamin B12 (cobalamin or methylcobalamin), vitamin C, vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B8 (inositol), vitamin B9 (folic acid), vitamin B12 (cobalamin or methylcobalamin), vitamin C, vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B8 (inositol), vitamin B9 (folic acid), vitamin B12 (cobalamin or methylcobalamin), vitamin C, vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), vitamin B6 (pyridoxine), vitamin B6 (biotin ...pyridoxine), vitamin B6 (biotin), vitamin B7 (biotin), vitamin B7 (biotin), vitamin B7 (biotin), vitamin B Vitamin D4 (22-dihydroergocalciferol), vitamin D5 (sitocalciferol), vitamin D6 (calciferol), vitamin D7, vitamin E, vitamin K1 (phylloquinone), vitamin K10 (phylloquinone epoxide), vitamin K2 (menaquinone), vitamin K3 (menadione), any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0182] In certain embodiments of the membrane-binding chemical fragment, amino acids and peptides and derivatives (AAPs) include, but are not limited to, glutamic acid (Glu), glutamine (Gln), glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), aspartic acid (Asp), histidine (His), asparagine (Asn), lysine (Lys), methionine (Met), arginine (Arg), serine (Ser), threonine (Thr), cysteine ​​(Cys), proline (Pro), peptides containing 2 to 60 amino acids, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0183] In certain embodiments of the membrane-binding chemical fragment, sugars and their derivatives (SCAs) include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides, glycosides, glycoproteins, glycolipids.

[0184] In certain embodiments of the membrane-binding chemical fragments, sugars and derivatives (SCAs) include, but are not limited to, D-ribose, L-ribose, D-arabinose, L-arabinose, D-xylose, L-xylose, D-lyxose, L-lyxose, D-allose, L-allose, D-altrose, L-altrose, D-glucose, L-glucose, D-mannose, L-mannose, D-gulose, L-gulose, D-idose ( ldose), L-idose, D-galactose, L-galactose, D-talose, L-talose, D-fucose, L-fucose, N-acetylneuraminic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, trehalose, maltose, sucrose, cellobiose, kestose, raffinose, nystose, fructosyl-nistose, glucan, arabinoxylan, apigenin-7-O-glucose Glycosides, quercetin-3-O-glucoside, isorhamnetin-3-O-rutinoside, kaempferol-p-coumaroylrhamnoside, quercetin-3-O-rutinoside, quercetin-3-O-glucuronide, kaempferol-3-O-rutinoside, isorhamnetin-O-pentoside, quercetin-3-O-rhamnoside, isorhamnetin-O-glucuronide, kaempferol-methyl ether-O-glucoside, isorhamnetin-O-acetylrutinoside, rhamnetin-O-glucuronide, quercetin-dimethyl ether-O-rutinoside, quercetin-dimethyl Methyl ether-O-glucuronide, kaempferol-O-p-coumaroyl rhamnoside, quercetin-7-O-glucoside, luteolin 7-O-glucoside, naringenin 7-rhamnoside, cerebroside, ganglioside, glucosyl cerebroside, lactosylceramide, hopane glycolipid, oligosaccharide or polysaccharide containing 2-100 monosaccharide and / or derivative units and which may be linear or branched, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0185] In certain embodiments of the membrane-bound chemical fragments, nucleobases and nucleoside and nucleotide derivatives (NNNA) include, but are not limited to, adenine, guanine, thymine, cytosine, uracil, hypoxanthine, xanthine, epiguanine, dihydrouracil, adenosine, guanosine, thymidine, cytidine, uridine, inosine, xanthine, 7-methylguanosine, dihydrouridine, cAMP, pppGpp, NADP, FAD, ATP, ADP, AMP, GTP, GDP, GMP, UTP, UDP, UMP, CTP, CDP, CMP, TTP, TDP, TMP, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0186] In certain embodiments, the reported membrane protein binding agents and derivatives (MPBDs) include, but are not limited to, atoms, drugs, portions or derivatives of drugs and chelates, any deuterium-substituted derivatives, pharmaceutically acceptable salts or stereoisomers, or any combination thereof, that target membrane components that mediate endocytosis.

[0187] In certain embodiments, chelating atoms include but are not limited to Ag. + 、Cu + 、Au + 、Hg 2+ , Pb 2+ 、Cu 2+ 、Cd 2+ 、Zn 2 + 、Se 2- 、Se 4+ 、Se 6+ and Sb 3+ .

[0188] In certain embodiments, the selected drug, a portion of a drug or a drug derivative and chelate (ADD), any deuterated derivative, pharmaceutically acceptable salt or stereoisomer or any combination thereof is that of a reported target protein. In certain embodiments, the target protein includes but is not limited to the target proteins listed in patent US20210002296A1, including all variants, mutations, splice variants, insertions and deletions and fusions of these target proteins listed. Examples include but are not limited to 5HT2c receptor, α 1A-AR, alpha-2 adrenergic receptor, alpha-synuclein, AAK1, ATP-binding cassette (ABC) transporters (such as MDR1 / 2 / 3 / 4 / 5 and ABCG2), ABL, cholesterol acyltransferase (ACAT), ACE protein, ACK, ACTR2, ACTR2B, ADCK1 / 2 / 3 / 4 / 5, protein kinase B (AKT), alphaK1 / 2 / 3, amino acid transporter, ALIX, anaplastic lymphoma kinase (ALK), anamniotic fluid protein, AMPA receptor (AMPAR), AMP-related protein kinase (AMPK), amyloid beta, angiotensin II receptor, ANKRD3, Apaf-1, apolipoprotein E receptor (ApoER), amyloid precursor protein (APP), androgen receptor (AR), ARAF, ARG, AR-V7, ileal apical sodium / bile acid cotransporter (ASBT), V-type proton ATPase 6 (ATP6V), A TP6V1H, avidin, alanine serine cysteine ​​transporter (ASCT), ASGPR, ASK1 / 2, ataxia-1, ataxia telangiectasia mutated protein (ATM), ATM and Rad3-related protein (ATR), Aurora kinase, AXL, beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), beta2-adrenergic receptor, BAD, BARK1 / 2, Bax, BCKDK, B cell leukemia / lymphoma (BCL) family proteins (such as BCL2, BCL-XL and MCL-1), BCR-ABL, bromodomain and extraterminal domain family proteins (BET) (such as BRD2 / 3 / 4 / T), beta-catenin, BIKE / BMP2K, BLK, BMPR1A, BMPR1B, BMPR2, BMX / ETX, CDO brothers (Boc), BRD9, BMI1, BRAF, BRAF V600E, brassinosteroid insensitive 1 (BRI1), BRK, BRM, BRSK1, BRSK2, Bruton's tyrosine kinase (BTK), BUB1, C3G, calreticulin, cell adhesion molecule (CAM) receptors, CAMK1α / β / γ / δ, CAMK2α / β / γ / δ, CAMK4, caMLCK, cannabinoid-1 receptor, coxsackievirus-adenovirus receptor (CAR), CRISPR-associated protein (Cas), CASK, caspase-3, caspase-6, caspase-7, caspase-9, CBFβ, CBL-B, CBP, chemokine receptors such as CC chemokine receptor (CCR) and CXC chemokine receptor ( CXCRs), such as CXCR2, CXCR4, and CXCR7), CCK4 / PTK7, CCR2, CCR9, CCRK, clusters of differentiation (CDs), such as CD4, CD9, CD13 / APN receptor, CD25, CD26 (DPP4), CD30, CD33, CD36, CD38, CD46, CD56, CD63, CD71, CD74, CD80, CD81, CD82, CD86, CD123, CD138, CD147, CD152, CD152 (CTLA4), CD166, CD174, CD197, CD205, CD227, CD228, CD269, CD276, and C D326), CDC7, CDC20, CDC25, CDC37, CDK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 / 16 / 17 / 18, CDKL1 / 2 / 3 / 4 / 5, CDON, CEACAM5, cholesteryl ester transfer protein (CETP), c-Fos, cystic fibrosis transmembrane conductance regulator (CFTR), cyclic GMP-AMP synthase (cGas), Chak1 / 2, CHK1 / 2, CI-M6PR, CK1α, CK1α2, CK1δ, CK1ε, CK1γ1 / 2 / 3, CK2α1 / 2, CLIK1, CLIK1L, Clip, CLK1 / 2 / 3 / 4, human collagen lectin placenta 1 (CL-P1), CMYC, cone opsin, COT / TPL2, cell penetrating peptide (CPP), junction protein, coronavirus protease, CRABP, C-RAF, cerebellum protein (CRBN), CREB, CRK, CRIK, CRIPTO, CSF2R, CSF1R / FMS, CSK, C-TAK1, CTK, pinocytosis receptor, cyclin D, cyclin E, CytoC, DAPK1 / 2 / 3, DCAMKL1 / 2 / 3, DDR1 / 2, diacylglycerol acyltransferase (DGAT), DLK, DMPK1 / 2, DNA-PK, DP-1, DPP-4, DRAK1 / 2,Deubiquitinating enzyme (DUB), DYRK1A / 1B / / 2 / 3 / 4, E2F, excitatory amino acid carrier 1 (EAAC1), E-cadherin, endothelin converting enzyme (ECE), ED-B, EED, EF2K, eEF2K, EIF2A3, EIF4E, EIF4G, ELK, ENL, endothelin B receptor, epithelial cell adhesion molecule (EpCAM), endothelial cell protein C receptor (EPCR), ephrin receptor (EphR), estrogen receptor (ER), ER81, ErbB2, ErbB3, ETS transcription factor (ERG), ERK1 / 2 / 3 / 4 / 5 / 7 / 8, ERRa, EZH2, fatty acid binding protein (FABP), FADD, FAK, FAP, fatty acid transport protein (FATP), neonatal Fc receptor (FCRN), FER, FKBP, FLIP, futurin-1, futurin-2, folate receptors (such as reduced folate carrier, FOLT1 / 2 / 3 and proton-coupled folate transporter (PCFT)), FOXM1, FOXO1 / 2 / 3 / 4, FRK, Frizzled4, fructose-1,6-bisphosphatase, FYN, gamma-aminobutyric acid type A receptor (GABAAR), GAK, Gap-1, growth arrest-specific 1 (Gas1), GCC, GCK, GCN2, GD2, ghrelin receptor (ghrelinR), glucose-dependent insulinotropic polypeptide (GIP), Globo H, glucagon receptor, glucagon-like peptide 1 (GLP-1), glutamate transporter (GLT), glucokinase, ionotropic AMPA glutamate receptor (GluR), glucose-6-phosphatase, glucose transporter (GLUT), glutathione transporter, glycogen phosphorylase, glycogen synthase kinase, glycoproteins (such as gp18, gp31 and gp60), G protein-coupled receptors (GPCRs) (such as GPR20, free fatty acid receptor 1 (GPR40), GPR119 and GPR120), GPN MB, GPRK4 / 5 / 6 / 7, GRB2, GSK-3, GSPT1, Haspin kinase, HCK, hepatitis C virus (HCV) NS3 / 4A, histone deacetylase (HDAC), HDL receptor (HDLR), HectH9, HH498, HIF1, HIPK1 / 2 / 3 / 4, HMGN1, HMG-CoA reductase, hnRNP, H-PGDS, HPGCR, hematopoietic progenitor cell kinase 1 (HPK1), HRAS, HRI, hRpn13, Pru, HSD-11β (11β-hydroxysteroid dehydrogenase), heat shock protein (HSP), huntingtin (HTT), HUNK, inhibitor of apoptosis protein (IAP) (such as cIAP and XIAP), ICK, intermediate density lipoprotein receptor (IDLR), IDO-1, IFN-γR, IGF-1R, IGF-2r, IKK-α, IKK-β, IKK-γ, IKK-ε, Ikaros (IKZF1), Helios ( IKZF2), Aiolos (IKZF3), IKZF4, IL-4R, IL-10R, ILK, integrins (such as αVβ3, α4β1, and α5β1 integrins), insulin receptor (IR), insulin-like growth factor receptor, INSR, IRE1 / 2, IRR, ITKIMP, integrin receptor (IR), IRA2, IRAK1 / 2 / 3 / 4, IRE1, Janus tyrosine kinase (JAK), c-Jun N-terminal kinase (JNK), potassium-chloride cotransporter 2 (KCC2), KDM5C, KHS1 / 2, inwardly rectifier potassium channel (Kir2.3), KIS, KIT, Kelch domain-containing protein 2 (KLHDC2), KLF4, KRAS, KRAS G12C, KSR1 / 2, lamin, lysosomal-associated membrane protein (Lamp), LAMTOR2, LANA, lactoferrin receptor, L-amino acid transporter (LAT), LATS1 / 2, LCK, low-density lipoprotein receptor (LDLR), LEF, LIMK1 / 2, LKB1, LMR1 / AATK, LMR2 / 3, LOK, low-density lipoprotein receptor-related protein (LRP), LRRK1 / 2, LTK, LXR-β, LYN, LZK, LZTFL1, m5C, m6A, MAK, MALT, MAP3K4 / 8, MAP4K3, MAP4K5, MAPK, MAPKlb, MAPKAPK2 / 3 / 5, MARK1 / 2 / 3 / 4, MAST1 / 2 / 3 / 4 / L, MC4 receptor, MCAK, MCH-1 receptor, monocarboxylate transporter (MCT), mouse double minute 2 homolog (MDM2) , MDMx, megalin, mitogen-activated protein kinase (MEK), MEKK1 / 2 / 3, MEL, MELK, MER, MERTK, mesothelin, MET, Mfsd2a, metabotropic glutamate receptor (mGlu1), major histocompatibility complex class I protein (MHC-I), MHC-II, MIF, MINCLE, MINK1, MISR2, MITF, MKK1 / 2 / 3 / 4 / 6 / 7, MKP3, MLK1 / 2 / 3 / 4, MLKL, MLN1 / 3, MNK1 / 2, MOK, MOS, MPK, MPSK1, MRCKα / β, multidrug resistance protein (MRP), MSK1 / 2, MSSK1, MST1 / 2 / 3 / 4, mammalian target of rapamycin (mTOR), MUCL, MUSK, Myc protein, MYO3A / 3B, MYT1, NAMPT, N-cadherin, K + Dependent Na + / Ca 2+exchanger 2 (NCKX2), NDC80, NDR1 / 2, NEK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11, NF-κB, nicotinic acetylcholine receptor, NIK, NLK, NOTCH receptor, Niemann-Pick C1-like 1 (NPC1L1), N-methyl D-aspartate receptor (NR), NRAS, NRBP1 / 2, NRF2, NSD2, NSD3, nucleoside transporters (NT, such as hENT1, hCNT1-3, etc.), sodium / taurocholate cotransporter transport peptide (NTCP), NuaK1 / 2, NUAK1, organic anion transporter (OAT), organic anion transporting polypeptide (OATP, OATP1B1, OATP2B1, OATP4C1, etc.), obscurin, organic cation transporter (OCT), OSR1, organic solute transporter (OST), otoferlin, P2X purinergic receptor 4 (P2x4R), purinergic receptor P2Y12 (P2Y12), p16INK4A, p18INK4, p19INK4D, p21, p27 kip1, p38α, p38β, p38γ, p38δ, p53, p65 / RELA, p70S6K, p70S6Kb, p90, p300 protein, PAK1 / 2 / 4 / 5 / 6, pan-coronavirus antiviral agent, protease-activated receptor (PAR), PARP, PASK, PAX, PBRM1, PCSK9, PD-1, phosphodiesterase (PDE), PDE4, PDEδ, PDGFRα / β, pdhk1 / 2 / 3 / 4, PDK1, PD-L1, PEA-15, PEIZO1, PEIZO2, PEK, peptide transporter (PEPT), P-glycoprotein (P-gp), PHKy1 / 2, phosphoenol Pyruvate carboxykinase, phosphoinositide 3-kinase (PI3K), PIM1 / 2 / 3, PINK1, PITSLRE, PKA, PKACα / β / γ, PKBα / β, PKC, PKD1 / 2 / 3, PKG1 / 2, PKN1 / 2 / 3, PKR, PLC, PLDL, PLK1 / 2 / 3 / 4, PMEL17, PRAK, peroxisome proliferator-activated receptor (PPAR), PRC2, PRK2, PRKX, PRKY, PRMT5, protein phosphatase, protein tyrosine phosphatase (PTP), PRP4, PRPK, PSKH1 / 2, prostate-specific membrane antigen (PSMA), receptor patched 1 (PTCH1), PTEN, protein tyrosine kinase (PTK), PVRL4, pyruvate dehydrogenase kinase, PYK2, QIK, QSK, R1P1, RACK-1, Rac protein, RAF protein, RAP1, Raptor, RAR, Ras protein family (RAS), Rb, retinol binding protein (RBP), RET, riboflavin transporter protein (RFVT), RFX1, RHAU, RHODK, RIOK1 / 2, receptor interacting protein kinase (RIPK), rhodopsin, ribonuclease K (RNASEK), RNAseL, RING finger protein (RNF), ROCK1 / 2, renal outer medullary potassium channel (ROMK), RON, ROR1 / 2, ROS, RPN11, RPN13, ribosomal s6 kinase, receptor serine / threonine kinases (RSKs) such as transforming growth factor β (TGF-β) receptor and Aristidis Moustakas et al., proteins listed in Receptor Serine / Threonine Kinases (ISBN: 978-3-540-44244-8), Receptor Tyrosine Kinases (RTKs) (such as fibroblast growth factor receptor (FGFR), FLT1 / 2 / 3 / 4, FGFR1 / 2 / 3 / 4, RET, tropomyosin-related kinase (TRK), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER), vascular endothelial growth factor receptor (VEGFR),TYRO3 and other proteins listed in the publication Mark A. Lemmon and Joseph Schlessinger, Cell, 141(7)1117-1134, 2010), RUNX1 / 2 / 3, RYK, spindle assembly checkpoint (SAC), Sanpodo / Notch, SAP, SARS-CoV-2, SBK, solute carrier (SC), SCF, SCYL1 / 2 / 3, SF3B1, serine / threonine protein kinase (SGK), SGLT, SHC, SHP2, SIK2 / 3, SIRT2, SIX1, skMLCK, S phase kinase-associated protein 1 (SKP1), S phase kinase-associated protein 2 (SKP2), solute carriers (SLC, such as SCL19A1) transporters (such as hMATE1 and as in the publication Enrico Girardi et al., Nature Chemical Biology, 16, 469-478, 2020), SLK, SLOB, Smac, Smad proteins, SMARCA, sodium-coupled monocarboxylate transporter (SMCT), SMG1, smMLCK, sodium-dependent multivitamin transporter (SMVT), synaptosomal-associated protein (SNAP), SNRK, sortilin-related CNS-expressed 1a (SorCS1a), SorCS1c, son of seven (SOS), SOS1, SOX2, secreted protein acidic and rich in cysteine ​​(SPARC), SPEG, speckled BTB / POZ protein (SPOP), SPRED, SPRY, SPv-NK1R, scavenger receptors (SR) (such as CD36, LAMP1 and LAMP2), Src proteins, SRF, SRM, SRPK1 / 2, SSTK, StaO, signal transducers and activators of transcription (STAT), STING, serine / threonine kinase enzyme (STK), STLK3 / 5 / 6, syntaxin (STX), SuRTK106, SUV39HI, SUZ12, sodium-vitamin C cotransporter (SVCT), synaptotagmin (SYT), triiodothyronine (T3), TAB, TAK1, TAL, TAO1 / 2 / 3, tau protein, TBCK, TBK1, T cell factor / lymphoid enhancer binding factor (TCF / LEF family), TEC, TESK1 / 2, TFAM, transcription factor EB (TFEB), transferrin receptor (TfR), TGF-β, trans Golgi network (TGN38), thiamine transporter (THTR), TIE1, TIE2 / TEK, TIF1a / β / γ, titin / TTN, TLK1 / 2, toll-like receptors (TLRs) (such as TLR4), TNK1, TOPK, TPL2 / COT, TPN1p, TRAD, TRAF proteins, Trb1 / 2 / 3,Tripartite motif family proteins (TRIM), Trio, TRRAP, TSC2, TSG101, TSSK1 / 2 / 3 / 4, TTBK1 / 2, TTK, TXK, TYK2, tyrosinase, tubulin, UBF, UCHL5, ULK1 / 2 / 3 / 4, ubiquitin-specific peptidase (USP) (such as USP7, USP11 and USP14), VACAMKL protein, vesicle-associated membrane protein (VAMP), vitamin D receptor (VDR), vesicular GABA transporter (VGAT), vesicular glutamate Transporter (VGLUT), very low density lipoprotein receptor (VLDLR), urate transporter 1 (URAT1), VRK1 / 2 / 3, WAVE-2, WDR5, WEE-1, WEE-1B, WNK1 / 2 / 3 / 4, Wnt, XBP1, YANK1 / 2 / 3, YBX1, YES, YSK1, ZAK, ZAP70, ZC1 / 2 / 3 / 4, ZFP91 and zinc ring finger protein (ZRNF), including all variants, mutations, splice variants, indels and fusions of these target proteins listed.

[0189] In certain embodiments, the multivalent endocytic agent is a bivalent or multivalent agent for protein inhibition and chemical modification of proteins, DNA or RNA, including but not limited to protein ubiquitination, protein deubiquitination, protein phosphorylation, protein methylation, protein acetylation, protein folding or unfolding, and DNA / RNA degradation.

[0190] In certain embodiments, bivalent or multivalent agents are protein degraders, including but not limited to protein degradation targeting chimeras (PROTACs). The PROTAC degradation agent of the protein after protein ubiquitination is a compound that physically degrades the target protein by proteolytic cleavage of one or more bonds of the target protein. PROTAC also includes all PROTAC variants, such as light cages (photocaged) PROTAC, label-based PROTACs, etc. Exemplary protein degraders with other mechanisms include but are not limited to protein molecule degraders, autophagy system linking compounds (ATTEC), autophagy targeting chimeras (AUTAC), chaperone-mediated protein degraders (CHAMPs), BacPROTACs, ASGPR targeting chimeras (ATACs), lysosomal targeting chimeras (LYTACs), mitochondrial protease targeting chimeras (MtPTACs). Bivalent or multivalent agents also include deubiquitinase targeting chimeras (DUBTACs), RESTORACs, enhancer targeting chimeras (ENTACs), and phosphorylation-inducing chimeric small molecules (PHICS), ribonuclease targeting chimeras (RIBOTACs), phosphatase recruitment chimeras (PhoRCs), dephosphorylation targeting chimeras (DEPTACs), phosphorylation targeting chimeras (PhosTACs), phosphorylation-inducing chimeric small molecules (PHICS), acetylation tagging molecules (AceTAGs), regulatory-inducible proximity targeting chimeras (RIPTACs), transcriptional / epigenetic chemical proximity inducers (TCIPs), caspase cleavage targeting chimeras (CACTACs), deuterated derivatives thereof, analogs thereof, or combinations thereof.

[0191] In certain embodiments, the functional moiety in the bivalent or multivalent agent or the bivalent or multivalent agent itself is represented by any of the following compounds: 3-aminophthalic acid (CAS: 5434-20-8), 4-aminoisoindoline-1,3-dione (CAS: 2518-24-3), 4-aminoisobenzofuran-1,3-dione (CAS: 17395-99-2), 5-aminoisoindoline-1,3-dione (CAS: 3676-85-5), 5-aminoisobenzofuran-1,3-diketone (CAS: 17011-53-9), 4-aminophthalic acid (CAS: 5434-21-9), 753b, A7, A16, A031, A1874 (CAS: 2064292-12-0), ABBV-101, Ab-PROTAC3, ACBI1 (CAS: 2375564-55-7), ACBI3, AC-0682, AC-0716, AC-0676, ACBI2 (CAS: 2913161-19-8), AGB1, AM-A3, AMG232 (CAS: 1352066-68-2), AP-01-104, APG-265, APR-Cy3, AR-VHL-SF2, AR2-VHL-SF2, ARCC-4 (CAS: 1973403-00-7), ARD-69 (CAS: 2316837-10-0), ARD-61 (CAS: 2316837-08-6), ARD-69, ARD-2128 (CAS: 2222111-87-5), ARD1676, GT20029, ARD-266, ARD-2051 (CAS: 2632305-17-8), ARD-2585 (CAS: 2757422-79-8), AR-ARL, AR-LDD, ARV110 (CAS: 2222112-77-6), ARV-766 (CAS: 2750830-09-0), ARV-471 (CAS: 2229711-68-4), ARV-771 (CAS: 1949837-12-0), ARV-825 (1818885-28-7), ARV-763, ASP-3082, AT-1 (CAS: 2098836-45-2), ATTEC, AU-15330, AUTAC4, Avadomide, PROTAC based on AZD9496 (CN112979747A), Azo-PROTAC-4C, β-NF-ATRA, BC5P, BCPyr, BETd-260 (CAS: 2093388-62-4), B03, BGB-166, BI-3663 (CAS: 2341740-84-7), BI3802 (CAS: 2166387-65-9), BP3, BRD4-SF2, BSJ-4-116 (CAS: 2519823-34-6), BT1, BTX-1188, BTX-9341, BWA-522, C004019, C13 (Jingyu Zhang et al., Journal of Medicinal Chemistry, 65, 13, 9096-9125,2022), CC885 (CAS: 1010100-07-8), CC-90009 (CAS: 1860875-51-9), CC-92480 (CAS: 2259648-80-9) (Joshua D. Hansen et al., Journal of Medicinal Chemistry,63,6648-6676,2020), CC-94676, CC-99282, CCR9-PROTAC, CCT369260, CCW 28-3, CDDO-JQ1, CDDO-Me, CFT7455 (NCT04756726), CFT8634 (CAS: 2704617-96-7), CFT8919, CFT1946 (CAS: 2882165-79-7), CG416, CG428, CG001419, CL1-YL2, CLL1-5, CM11, compound 3 (Mingming Wei et al., European Journal of Medicinal Chemistry, 209, 112903, 2021), compound 6 (Archana Bhumireddy et al., Bioorganic & Medicinal Chemistry Letter, 55, 128448, 2022), compound 6c (Lijie Peng et al., ACS Medicinal Chemistry Letters, 10, 767-772, 2019), compound 9 (Joao Nunes et al., ACS Medicinal Chemistry Letters, 10, 1081-1085, 2019), compound 21b (Guoshun Luo et al., ActaPharmaceutica Sinica B, 11(5), 1300-1314, 2021), compound 955 (Jing Pei et al., CellChemical Biology, 30, 203-213, 2023), CP5V (CAS: 2509359-75-3), CP-10 (CAS: 2366268-80-4), CPD-1224 (CAS: 2891620-68-9), CPR3, CPR4, CPS2, CRBN-6-5-VHL, D15 (Pengyun Li et al. Acta Pharmaceutica Sinica B,13(6),2715-2735,2023), d4E-4, d4E-6, d9A-2, dBET1(CAS:1799711-21-9), dBET6(CAS:1950634-92-0), DBt-10(Martin,etc., bioRxiv preprint, 2023, DOI: 10.1101 / 2023.04.09.536153), dCBP-1 (CAS: 2484739-25-3), DCY-09-192, DD-03-171 (CAS: 2366132-45-6), DD-04-015, dFKBP-1 (CAS: 1799711-22-0), DGY-04-035, DGY-06-177, DGY-06-177-pk1, DGY-06-177-pk2, DGY-09-192, DKY709 (NCT03891953), dMCL1-2 (CAS: 2351218-88-5), DP1, DP-C-1, DP-C-4, DP-V-4, DS-3032 (CAS: 1398568-47-2), DT2216 (CAS: 2365172-42-3), dTRIM24 (CAS: 2170695-14-2), ERD-148, ERD-308 (CAS: 2320561-35-9), ERD-3111 (CAS: 2832865-25-3), ERG OP-C1, ER PROTAC (ES2717436T3), FA-S2-POMA, FA-S2-MS4048, FHD-609 (CAS: 2676211-64-4), folic acid-ARV-771, folic acid-MS432, folic acid-MS99, Fulvestrant, G4-PROTAC, GBD-9, GBM-475, GMB-475 (CAS: 2490599-18-1), GNE-987, GSK215, GT20029, GT19630, GT19715, GW3965-PEG5-VH032, HaloPROTAC-3, HBL-4, HC-X029, HC-X035, HD-TAC7, HJM-561, HER2-14, HP14, HP17, HP518, HP568, HPB-143, HRS-1358, HRS-5041, HSK29116, I-6, I-685, Iberdomide, INY-03-041, ITRI-90, ITRI-125 and ITRI-126 (Chiu-Lien Hung et al., eBioMedicine, 90, 104500,2023), JB170, JH-XI-10-02 (CAS: 2209085-22-1), JP-1, JP-2, JP-3, JP-4, JP-5, JP-6, JPX-0802, JPX-1185, JPX1188, JNJ-1013, JMKX000623, KB02-JQ-1 (CAS: 2384184-44-3), KB02-SLF (CAS: 2384184-40-9), KP-14, KRAS PROTAC (patent WO2022173032, including KRAS G12D inhibitor 17 (CAS: 2821793-99-9)), KT-333, KT-413, KT-253, KT-474 (CAS: 2432994-31- 3), KTX-335, KTX-652, KTX-959, KTX-978, KTX-214, KYH1872, L18i, LC-2, LC-BM12, LEF1 OP-V1, LC-MB12 (CAS: 828438-38-4), lenalidomide, LG1188, LT-002, macroPROTAC-1, MK-8242 (CAS: 147-94-4), MD13, MD-222 (CAS: 2136246-72-3), MD-224 (CAS: 2136247-12-4), MDEG-541, MEK PROTAC3 (Stefan Vollmer et al., Journal of Medicinal Chemistry, 63, 157-162, 2020), MG-277 (CAS: 2411085-89-5), MM-02-08, MM-03-75 and MM-04-09 (Margot Meyers et al., bioRxiv Preprint, 2023,DOI:10.1101 / 2023.08.11.553046), MS-170(CAS:2376136-61-5), MR837(CAS:1210 906-48-1), MS28(CAS:2093386-22-0), MS33, MS39, MS67, MS83, MS98, MS154, MS170, M S4077(CAS:2230077-10-6), MS432, MS910, MS928, MS934, MS1943, MS4332, MS9715, MT -802(CAS:2231744-29-7), MTX-23(CAS:2488296-74-6), MZ1, N3-NF-κB-ODN, dNF-κB 15, dNF-κB 16, N3-E2F-ODN, dE2F 16, dE2F 17, NH2, NJH-04-086, NJH-04-087, NJH-04-098, NR-6a, NR-7h, Nutlin 3a, Nutlin 3, NVP-CGM097 (CAS: 1313363-54-0), NRX-0492 (CAS: 2416130-57-7), NX-0479, NX-2127 (CAS: 2416131-46-7), NX-5948 (CAS: 2649400-34-8), obrutinib, ORM-5029, ovalicin, P3, P4B, P19A s, P19P, P22, P22A, P22D, PAP508, pc-PROTAC1, PG21, pc-PROTAC3, P13i (CAS:2360561-66-4), PF15, pomalidomide (pomalidomide), PP-C8, precursor PROTAC, PROTAC-8, PROTAC_ERRα(CAS:1801547-15-8), PROTAC_RIPK2(Daniel P.Bondeson et al., Nature Chemical Biology, 11, 611-617, 2015), RIPK2 PROTAC (Anh-Tuan Pham et al., Frontiers in Pharmacology, 14, 1127722,2023), PRE3789, PROTAC MDM2 degrader-1 (CAS: 2249944-98-5), PROTAC BRD9 degrader-1 (CAS: 2097971-01-0), PROTAC BET degrader 23, PROTAC-D, PROTAC ER degrader-3 (CAS: 2158322-29-1), PROTAC-FCPF, PROTAC-O412, PROTAC (HPGDS) -1, PROTAC (HPGDS) -7, PRTC, PZ703b, PZ15527, QCA570 (CAS: 2207569-08-0), R1-5C, RBN012811, RC-1, RC-3, RG7112 (CAS: 939981-39-2), RG7388 (CAS: 1229705- 06-9), RNK05047, Rucaparib-AP6, SAR405838 (CAS: 1303607-60-4), SARD279, SD-36 (CAS: 2429877-44-9), SD-91, SHP2-D26 (CAS: 2458219-65-1), SIAIS001, SIAIS056, SIAIS091, SIAIS117, SIAIS164018, SIAIS117 (CAS: 2353 494-84-3), SIAIS125, SIAIS126, SIAIS178 (2376047-73-1), SIAIS629048, SIAIS629049, SIAIS629050, SIAIS629051 ,SIM1,SJ995973(CAS:2882065-25-8),SJF620(CAS:2376187-16-3),SJF-0628,SJF-0661,SJH1-62B,SK-575,SNIPER( ER)-3, SNIPER (ER)-87 (CAS: 2222354-91-6), SNIPER-BRD4-1, SNIPER-ABL-62, SP4 (CAS: 2624181-69-5), SPB5208, SMD-3040, SR-1114, STEAP1-5a, STEAP1-13a, stimulus-responsive PROTAC (sr-PROTAC), toluenesulfonic acid T1101 (CAS: 2250404-95-4), TD-004 (Chung Hyo Kang et al., Biochemical and Biophysical Research Communications, 502 (2), 542-547,2018.), TD-9, TD-165, TD-428 (CAS: 2334525-50-5), TD-802, thalidomide, TAI-1 (CAS: 1334921-03-7), TM-P4-Thal, TMX-2172, UBX-382, UI-EP002, UNC6852 (CAS: 2688842-08-0), UNC7700, UNC7698, VHLL—X-BCN No. 15, VHLL—X-BCN No. 16, VHLL—X-BCN No. 17, Versortrexate (VSTX), VZ185, WB214, WWL0245, xStAx-VHLL, XD2-149, XY028-140 (CAS: 2229974-83-6), XY-06-007, XH2, XL01126, XL5-VHL-2, XY-4-88, XY-07-035, XY-07-096, XY-07-093, XY-07-143, XY-07-189, 002, YF135, YKL-04-085, YM181, YUM70(CAS:423145-35-1), YX-2-107, YX-02-030(Clare M. Adams et al., Cancer Discovery, 13(5), 1210-1229, 2023), YZ167, YZ268, ZB-S-29, ZCY-PROTAC, ZNL-02-096, ZXH-4-130 and ZXH-4-137, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier. ,

[0192] In certain embodiments, the bivalent or multivalent agent is a PROTAC molecule. PROTAC is a heterobifunctional molecule consisting of one or more warheads that bind to a target protein (POI), a linker, and one or more ligands that recruit E3 ubiquitin ligases. By simultaneously binding to the POI and the E3 ligase, PROTAC can bring two or more proteins into proximity and promote ubiquitination to achieve subsequent proteasomal degradation of the POI. In contrast to protein inhibition, the event-driven pharmacological mechanism of PROTAC is catalytic in nature. Concentrations below the effective inhibitory drug concentration will provide sufficient and lasting target degradation and avoid off-target toxicity caused by high-dose drugs. Unfortunately, PROTACs have poor durability due to their high molecular weight, which is generally associated with metabolic instability, poor solubility, and poor permeability. In theory, metabolic vulnerability and insolubility can be completely addressed by blocking metabolic hotspots and forming salts on basic or acidic functional groups, respectively. In addition, formulation techniques can be used to enhance water solubility. However, as demonstrated by a wealth of published evidence, passive penetration of PROTACs has been found to be difficult or impossible, and structural modification of PROTACs to achieve better cell permeability via classical medicinal chemistry has been significantly limited or even impossible due to the large molecular size of PROTAC agents.

[0193] The PROTAC warhead can target any suitable protein of interest (POI). In certain embodiments, the target protein includes but is not limited to the target proteins listed in patent US20210002296A1, including all variants, mutations, splice variants, insertions and deletions and fusions of these target proteins listed. Examples include but are not limited to 5HT2c receptor, α 1A-AR, alpha-2 adrenergic receptor, alpha-synuclein, AAK1, ATP-binding cassette (ABC) transporters (such as MDR1 / 2 / 3 / 4 / 5 and ABCG2), ABL, cholesterol acyltransferase (ACAT), ACE protein, ACK, ACTR2, ACTR2B, ADCK1 / 2 / 3 / 4 / 5, protein kinase B (AKT), alphaK1 / 2 / 3, amino acid transporter, anaplastic lymphoma kinase (ALK), ALIX, anamnion, AMPA receptor (AMPAR), AMP-related protein kinase (AMPK), amyloid beta, angiotensin II receptor, ANKRD3, Apaf-1, apolipoprotein E receptor (ApoER), amyloid precursor protein (APP), androgen receptor (AR), ARAF, ARG, AR-V7, ileal apical sodium / bile acid cotransporter (ASBT), V-type proton ATPase 6 (ATP6V), A TP6V1H, avidin, alanine serine cysteine ​​transporter (ASCT), ASGPR, ASK1 / 2, ataxia-1, ataxia telangiectasia mutated protein (ATM), ATM and Rad3-related protein (ATR), Aurora kinase, AXL, beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), beta2-adrenergic receptor, BAD, BARK1 / 2, Bax, BCKDK, B cell leukemia / lymphoma (BCL) family proteins (such as BCL2, BCL-XL and MCL-1), BCR-ABL, bromodomain and extraterminal domain family proteins (BET) (such as BRD2 / 3 / 4 / T), beta-catenin, BIKE / BMP2K, BLK, BMPR1A, BMPR1B, BMPR2, BMX / ETX, CDO brothers (Boc), BRD9, BMI1, BRAF, BRAF V600E, brassinosteroid insensitive 1 (BRI1), BRK, BRM, BRSK1, BRSK2, Bruton's tyrosine kinase (BTK), BUB1, C3G, calreticulin, cell adhesion molecule (CAM) receptors, CAMK1α / β / γ / δ, CAMK2α / β / γ / δ, CAMK4, caMLCK, cannabinoid-1 receptor, coxsackievirus-adenovirus receptor (CAR), CRISPR-associated protein (Cas), CASK, caspase-3, caspase-6, caspase-7, caspase-9, CBFβ, CBL-B, CBP, chemokine receptors such as CC chemokine receptor (CCR) and CXC chemokine receptor ( CXCRs), such as CXCR2, CXCR4, and CXCR7), CCK4 / PTK7, CCR2, CCR9, CCRK, clusters of differentiation (CDs), such as CD4, CD9, CD13 / APN receptor, CD25, CD26 (DPP4), CD30, CD33, CD36, CD38, CD46, CD56, CD63, CD71, CD74, CD80, CD81, CD83, CD86, CD123, CD138, CD147, CD152, CD152 (CTLA4), CD166, CD174, CD197, CD205, CD227, CD228, CD269, CD276, and C D326), CDC7, CDC20, CDC25, CDC37, CDK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 / 16 / 17 / 18, CDKL1 / 2 / 3 / 4 / 5, CDON, CEACAM5, cholesteryl ester transfer protein (CETP), c-Fos, cystic fibrosis transmembrane conductance regulator (CFTR), cyclic GMP-AMP synthase (cGas), Chak1 / 2, CHK1 / 2, CI-M6PR, CK1α, CK1α2, CK1δ, CK1ε, CK1γ1 / 2 / 3, CK2α1 / 2, CLIK1, CLIK1L, Clip, CLK1 / 2 / 3 / 4, human collagen lectin placenta 1 (CL-P1), CMYC, cone opsin, COT / TPL2, cell penetrating peptide (CPP), junction protein, coronavirus protease, CRABP, C-RAF, cerebellum protein (CRBN), CREB, CRK, CRIK, CRIPTO, CSF2R, CSF1R / FMS, CSK, C-TAK1, CTK, pinocytosis receptor, cyclin D, cyclin E, CytoC, DAPK1 / 2 / 3, DCAMKL1 / 2 / 3, DDR1 / 2, diacylglycerol acyltransferase (DGAT), DLK, DMPK1 / 2, DNA-PK, DP-1, DPP-4, DRAK1 / 2,Deubiquitinating enzyme (DUB), DYRK1A / 1B / / 2 / 3 / 4, E2F, excitatory amino acid carrier 1 (EAAC1), E-cadherin, endothelin converting enzyme (ECE), ED-B, EED, EF2K, eEF2K, EIF2A3, EIF4E, EIF4G, ELK, ENL, endothelin B receptor, epithelial cell adhesion molecule (EpCAM), endothelial cell protein C receptor (EPCR), ephrin receptor (EphR), estrogen receptor (ER), ER81, ErbB2, ErbB3, ETS transcription factor (ERG), ERK1 / 2 / 3 / 4 / 5 / 7 / 8, ERRa, EZH2, fatty acid binding protein (FABP), FADD, FAK, FAP, fatty acid transport protein (FATP), neonatal Fc receptor (FCRN), FER, FKBP, FLIP, futurin-1, futurin-2, folate receptors (such as reduced folate carrier, FOLT1 / 2 / 3 and proton-coupled folate transporter (PCFT)), FOXM1, FOXO1 / 2 / 3 / 4, FRK, Frizzled4, fructose-1,6-bisphosphatase, FYN, gamma-aminobutyric acid type A receptor (GABAAR), GAK, Gap-1, growth arrest-specific 1 (Gas1), GCC, GCK, GCN2, GD2, ghrelin receptor (ghrelinR), glucose-dependent insulinotropic polypeptide (GIP), Globo H, glucagon receptor, glucagon-like peptide 1 (GLP-1), glutamate transporter (GLT), glucokinase, ionotropic AMPA glutamate receptor (GluR), glucose-6-phosphatase, glucose transporter (GLUT), glutathione transporter, glycogen phosphorylase, glycogen synthase kinase, glycoproteins (such as gp18, gp31 and gp60), G protein-coupled receptors (GPCRs) (such as GPR20, free fatty acid receptor 1 (GPR40), GPR119 and GPR120), GPN MB, GPRK4 / 5 / 6 / 7, GRB2, GSK-3, GSPT1, Haspin kinase, HCK, hepatitis C virus (HCV) NS3 / 4A, histone deacetylase (HDAC), HDL receptor (HDLR), HectH9, HH498, HIF1, HIPK1 / 2 / 3 / 4, HMGN1, HMG-CoA reductase, hnRNP, H-PGDS, HPGCR, hematopoietic progenitor cell kinase 1 (HPK1), HRAS, HRI, hRpn13, Pru, HSD-11β (11β-hydroxysteroid dehydrogenase), heat shock protein (HSP), huntingtin (HTT), HUNK, inhibitor of apoptosis protein (IAP) (such as cIAP and XIAP), ICK, intermediate density lipoprotein receptor (IDLR), IDO-1, IFN-γR, IGF-1R, IGF-2r, IKK-α, IKK-β, IKK-γ, IKK-ε, Ikaros (IKZF1), Helios ( IKZF2), Aiolos (IKZF3), IKZF4, IL-4R, IL-10R, ILK, integrins (such as αVβ3, α4β1, and α5β1 integrins), insulin receptor (IR), INSR, IRE1 / 2, IRR, ITKIMP, integrin receptor (IR), insulin-like growth factor receptor, IRA2, IRAK1 / 2 / 3 / 4, IRE1, Janus tyrosine kinase (JAK), c-Jun N-terminal kinase (JNK), potassium-chloride cotransporter 2 (KCC2), KDM5C, KHS1 / 2, inwardly rectifier potassium channel (Kir2.3), KIS, KIT, Kelch domain-containing protein 2 (KLHDC2), KLF4, KRAS, KRAS G12C, KSR1 / 2, lamin, lysosomal-associated membrane protein (Lamp), LAMTOR2, LANA, lactoferrin receptor, L-amino acid transporter (LAT), LATS1 / 2, LCK, low-density lipoprotein receptor (LDLR), LEF, LIMK1 / 2, LKB1, LMR1 / AATK, LMR2 / 3, LOK, low-density lipoprotein receptor-related protein (LRP), LRRK1 / 2, LTK, LXR-β, LYN, LZK, LZTFL1, m5C, m6A, MAK, MALT, MAP3K4 / 8, MAP4K3, MAP4K5, MAPK, MAPKlb, MAPKAPK2 / 3 / 5, MARK1 / 2 / 3 / 4, MAST1 / 2 / 3 / 4 / L, MC4 receptor, MCAK, MCH-1 receptor, monocarboxylate transporter (MCT), mouse double minute 2 homolog (MDM2) , MDMx, megalin, mitogen-activated protein kinase (MEK), MEKK1 / 2 / 3, MEL, MELK, MER, MERTK, mesothelin, MET, metabotropic glutamate receptor (mGlu1), Mfsd2a, major histocompatibility complex class I protein (MHC-I), MHC-II, MIF, MINCLE, MINK1, MISR2, MITF, MKK1 / 2 / 3 / 4 / 6 / 7, MKP3, MLK1 / 2 / 3 / 4, MLKL, MLN1 / 3, MNK1 / 2, MOK, MOS, MPK, MPSK1, MRCKα / β, multidrug resistance protein (MRP), MSK1 / 2, MSSK1, MST1 / 2 / 3 / 4, mammalian target of rapamycin (mTOR), MUCL, MUSK, Myc protein, MYO3A / 3B, MYT1, NAMPT, N-cadherin, K + Dependent Na + / Ca 2+exchanger 2 (NCKX2), NDC80, NDR1 / 2, NEK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11, NF-κB, nicotinic acetylcholine receptor, NIK, NLK, NOTCH receptor, Niemann-Pick C1-like 1 (NPC1L1), N-methyl D-aspartate receptor (NR), NRAS, NRBP1 / 2, NRF2, NSD2, NSD3, nucleoside transporter (NT), sodium / taurine NTCP, NuaK1 / 2, NUAK1, organic anion transporter (OAT), organic anion transporting polypeptide (OATP), obscurin, organic cation transporter (OCT), OSR1, organic solute transporter (OST), otoferlin, P2X purinergic receptor 4 (P2x4R), purinergic receptor P2Y12 (P2Y12), p16INK4A, p18INK4, p19INK4D, p21, p27 kip1, p38α, p38β, p38γ, p38δ, p53, p65 / RELA, p70S6K, p70S6Kb, p90, p300 protein, PAK1 / 2 / 4 / 5 / 6, pan-coronavirus antiviral agent, protease-activated receptor (PAR), PARP, PASK, PAX, PBRM1, PCSK9, PD-1, phosphodiesterase (PDE), PDE4, PDEδ, PDGFRα / β, pdhk1 / 2 / 3 / 4, PDK1, PD-L1, PEA-15, PEK, peptide transporter (PEPT), P-glycoprotein (P-gp), PHKy1 / 2, phosphoenolpyruvate carboxykinase, Phosphoinositide 3-kinase (PI3K), PIM1 / 2 / 3, PINK1, PITSLRE, PKA, PKACα / β / γ, PKBα / β, PKC, PKD1 / 2 / 3, PKG1 / 2, PKN1 / 2 / 3, PKR, PLC, PLDL, PLK1 / 2 / 3 / 4, PMEL17, PRAK, peroxisome proliferator-activated receptor (PPAR), PRC2, PRK2, PRKX, PRKY, PRMT5, protein phosphatase, protein tyrosine phosphatase (PTP), PRP4, PRPK, PSKH1 / 2, prostate-specific membrane antigen (PSMA), receptor patched 1 (PTCH1), PTEN, protein tyrosine kinase (PTK), PVRL4, pyruvate dehydrogenase kinase, PYK2, QIK, QSK, R1P1, RACK-1, Rac protein, RAF protein, RAP1, Raptor, RAR, Ras protein family (RAS), Rb, retinol binding protein (RBP), RET, riboflavin transporter protein (RFVT), RFX1, RHAU, RHODK, RIOK1 / 2, receptor interacting protein kinase (RIPK), rhodopsin, ribonuclease K (RNASEK), RNAseL, RING finger protein (RNF), ROCK1 / 2, renal outer medullary potassium channel (ROMK), RON, ROR1 / 2, ROS, RPN11, RPN13, ribosomal s6 kinase, receptor serine / threonine kinases (RSKs) such as transforming growth factor β (TGF-β) receptor and Aristidis Moustakas et al., proteins listed in Receptor Serine / Threonine Kinases (ISBN: 978-3-540-44244-8), Receptor Tyrosine Kinases (RTKs) (such as fibroblast growth factor receptor (FGFR), FLT1 / 2 / 3 / 4, FGFR1 / 2 / 3 / 4, RET, tropomyosin-related kinase (TRK), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER), vascular endothelial growth factor receptor (VEGFR),TYRO3 and other proteins listed in the publication Mark A. Lemmon and Joseph Schlessinger, Cell, 141(7)1117-1134, 2010), RUNX1 / 2 / 3, RYK, spindle assembly checkpoint (SAC), Sanpodo / Notch, SAP, SARS-CoV-2, SBK, solute carrier (SC), SCF, SCYL1 / 2 / 3, SF3B1, serine / threonine protein kinase (SGK), SGLT, SHC, SHP2, SIK2 / 3, SIRT2, SIX1, skMLCK, S phase kinase-associated protein 1 (SKP1), S phase kinase-associated protein 2 (SKP2), solute carrier (SLC) transporters (such as hMATE1 and as in the publication Enrico Girardi et al., Nature Chemical Biology, 16, 469-478, 2020), SLK, SLOB, Smac, Smad proteins, SMARCA, sodium-coupled monocarboxylate transporter (SMCT), SMG1, smMLCK, sodium-dependent multivitamin transporter (SMVT), synaptosomal-associated protein (SNAP), SNRK, sortilin-related CNS-expressed 1a (SorCS1a), SorCS1c, son of seven (SOS), SOS1, SOX2, secreted protein acidic and rich in cysteine ​​(SPARC), SPEG, speckled BTB / POZ protein (SPOP), SPRED, SPRY, SPv-NK1R, scavenger receptors (SR) (such as CD36, LAMP1 and LAMP2), SR-B, Src proteins, SRF, SRM, SRPK1 / 2, SSTK, StaO, signal transducers and activators of transcription (STAT), STING, serine Acid / threonine kinase (STK), STLK3 / 5 / 6, syntaxin (STX), SuRTK106, SUV39HI, SUZ12, sodium-vitamin C cotransporter (SVCT), synaptotagmin (SYT), triiodothyronine (T3), TAB, TAK1, TAL, TAO1 / 2 / 3, tau protein, TBCK, TBK1, T cell factor / lymphoid enhancer binding factor (TCF / LEF family), TEC, TESK1 / 2, TFAM, transcription factor EB (TFEB), transferrin receptor (TfR), TGF-β, trans Golgi network (TGN38), thiamine transporter (THTR), TIE1, TIE2 / TEK, TIF1a / β / γ, titin / TTN, TLK1 / 2, toll-like receptors (TLR) (such as TLR4), TNK1, TOPK, TPL2 / COT, TPN1p, TRAD, TRAF proteins, Trb1 / 2 / 3,Tripartite motif family proteins (TRIM), Trio, TRRAP, TSC2, TSG101, TSSK1 / 2 / 3 / 4, TTBK1 / 2, TTK, TXK, TYK2, tyrosinase, tubulin, UBF, UCHL5, ULK1 / 2 / 3 / 4, ubiquitin-specific peptidase (USP) (such as USP7, USP11 and USP14), VACAMKL protein, vesicle-associated membrane protein (VAMP), vitamin D receptor (VDR), vesicular GABA transporter (VGAT), vesicular glutamate Transporter (VGLUT), very low density lipoprotein receptor (VLDLR), urate transporter 1 (URAT1), VRK1 / 2 / 3, WAVE-2, WDR5, WEE-1, WEE-1B, WNK1 / 2 / 3 / 4, Wnt, XBP1, YANK1 / 2 / 3, YBX1, YES, YSK1, ZAK, ZAP70, ZC1 / 2 / 3 / 4, ZFP91 and zinc ring finger protein (ZRNF), including all variants, mutations, splice variants, indels and fusions of these target proteins listed.

[0194] In certain embodiments, the E3 ligase is selected from, but not limited to, the following proteins: inhibitor of apoptosis (IAP), X-linked inhibitor of apoptosis (XIAP), aryl hydrocarbon receptor (AhR), RING finger protein 4 (RNF4), RING finger protein 114 (RNF114), Fem-1 homolog B (FEM1B), von Hippel–Lindau (VHL), casitas B-lineage lymphoma proto-oncogene-B (CBL-B) protein, cerebellin (CRBN), damage-specific DNA-binding protein 1 (DDB1), murine double minute 2 homolog (MDM2) protein, Kelch-like ECH-associated protein 1 (KEAP1), Kelch domain-containing protein 2 (KLHDC2), S phase kinase-associated protein 1 (SKP1), S phase kinase-associated protein 2 (SKP2), ubiquitin-protein ligase N-recognizer 5 (UBR5), DDB1 and CUL4-associated factor 1 (DCAF1) protein, DDB1 and CUL4-associated factor 11 (DCAF11) protein, DDB1 and CUL4-associated factor 15 (DCAF15) protein, and DDB1 and CUL4-associated factor 16 (DCAF16), including all variants, mutations, splice variants, indels, and fusions of these target proteins listed.

[0195] A PROTAC may comprise one or more than one E3 ligase ligand that targets IAP, VHL, CBL-B, CRBN, DDB1, MDM2, KEAP1, KLHDC2, SKP1, SKP2, DCAF1, DCAF15, DCAF16, UBR5, or any combination thereof.

[0196] POI binding ligand and E3 ligase ligand can be linked via chemical bonds or linker units Chemical connection or coupling. Chemical bond or linker unit Can be linked to any site of the POI binding ligand and E3 ligase ligand. The linker should allow for proper formation of the target protein-ligase complex. The linker group can contain one or more structural units.

[0197] In certain embodiments, the chemical bond or linker unit It can be a polyvalent polyethylene glycol, alkoxy, alkyl, alkylene, alkyne, cycloalkyl, cycloalkylene, cycloalkyne, heterocyclyl, aryl, heteroaryl, which can be terminated (at one or both ends) with at least one of the following: -H, -D, -O(R), =O, -S(R), =S, =NR, =N(R), -N(R)(R'), -Se(R), =Se, -F, -Cl, -Br, -I, -F, - 18F, -C(R)(R')(R”), =C(R)(R’), ≡C(R), -Si(R)(R’)(R”), =Si(R)(R’), ≡Si(R), -OS(O)2R, -OS(O)R, -S(O)R, -S(O)2R , -P(O)(R)(R'), -P(R)(R'), -P(O)(OH)O-, -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(S)O-, - OC(S)-, -OC(S)O-, -C(NOR)-, -C(O)N(R')-, -C(O)N(R)C(O)-, -C(O)N(R)C(O)N(R')-, -N(R)C(O)-, -N(R')C(O)N(R )-, -N(R)C(O)O-, -OC(O)N(R)-, -C(NR')-, -N(R)C(NR')-, -C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(O)2-, -OS(O)-, - S(O)O-, -S(O)-, -OS(O)2--S(O)20-, -N(R)S(O)2-, -S(O)2N(R)-, -N(R')S(O)-, -S(O)N(R)-, -N(R)S(O)2N(R')-, - N(R')S(O)N(R')-, -SS-, -O-Si(R)(R')-O-,)-, -C(=N)N(R)(R')-, -C(R)=C(R')-, -C≡C-, C3-12 cycloalkane, C3-12 cycloalkene, C3-12 cycloalkyne, a 3- to 12-membered heterocycle, a 5- to 12-membered aryl, a 5- to 12-membered heteroaryl, any deuterium-substituted derivative, or any combination thereof, wherein R, R', or R" is H, D, a 1-100 polyethylene glycol, a C1-C100 alkoxy, a C1-C100 alkyl, a C2-C100 alkylene, a C2-C100 alkyne, a C3-C100 cycloalkyl, a C3-C100 cycloalkylene, a C3-C100 cycloalkyne, a C3-C100 heterocyclyl, a C6-C100 aryl, or a C1-C100 heteroaryl, wherein one or two end-capping groups may be the same or different.

[0198] In certain embodiments, the chemical bond or linker unit Can be a multivalent chain unit It contains one or more than one chemical bond or linker unit Cores connected in any quantity and order wherein m and n represent integers from 0 to 100.

[0199] In certain embodiments, the core Including but not limited to atoms H, C, Si, N, P, B, O, S, Se, 1-100 polyethylene glycol, C1-C100 alkoxy, C1-C100 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkylene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl or C1-C100 heteroaryl, any deuterium substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0200] In certain embodiments, the chemical bond or linker unit It can be a divalent or trivalent polyethylene glycol, alkoxy, alkyl, alkylene, alkyne, cycloalkyl, cycloalkylene, cycloalkyne, heterocyclyl, aryl, heteroaryl, which can be terminated (at one or both ends) with at least one of the following: -H, -D, -O(R), =O, -S(R), =S, =NR, =N(R), -N(R)(R'), -Se(R), =Se, -F, -Cl, -Br, -I, -F, - 18- P(O)(R)(R'), -P(R)(R'), -P(O)(OH)O-, -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(S)O-, -OC(S) -, -OC(S)O-, -C(NOR)-, -C(O)N(R')-, -C(O)N(R)C(O)-, -C(O)N(R)C(O)N(R')-, -N(R)C(O)-, -N(R')C(O)N(R)-, -N(R )C(O)O-, -OC(O)N(R)-, -C(NR')-, -N(R)C(NR')-, -C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(O)2-, -OS(O)-, -S(O)O-, -S( O)-, -OS(O)2-, -S(O)2O-, -N(R)S(O)2-, -S(O)2N(R)-, -N(R')S(O)-, -S(O)N(R)-, -N(R)S(O)2N(R')-, -N(R')S(O)N(R')-, -SS-, -O-Si(R)(R')-O-,)-, -C(=N)N(R)(R')-, -C(R)=C(R')-, -C≡C-, C3-12 cycloalkane, C3-12 cycloalkene, C3-12 cycloalkyne, 3 to 12 membered heterocycle, 5 to 12 In some embodiments, m or n is an integer from 0 to 50.

[0201] In certain embodiments, PROTAC targets androgen receptor (AR), B-cell lymphoma-2 (BCL-2) family proteins, bromodomain and extraterminal (BET) family proteins, bromodomain-containing 9 (BRD9) protein, Bruton's tyrosine kinase (BTK), CREB binding protein (CBP) and / or p300 protein, cyclin-dependent kinase (CDK), epidermal growth factor receptor (EGFR), estrogen receptor (ER), interleukin-1 receptor-associated kinase (IRAK), Janus kinase (JAK) family proteins, Myc proteins, RAF proteins, RAS proteins, SMARCA proteins, signal transducers and activators of transcription (STAT), tau proteins, tropomyosin receptor kinase (TRK), or any combination thereof.

[0202] In certain embodiments, the bivalent or multivalent agent is an ATAC or LYTAC. ATAC or LYTAC is a heterobifunctional molecule consisting of a POI ligand, an endocytosis agent, and a linker between the two moieties. ATAC or LYTAC can trigger lysosomal degradation of membrane POIs or extracellular POIs. In certain embodiments, the ATAC or LYTAC herein, or the moiety within a LYTAC, is an agent that binds to membrane components that mediate endocytosis (such as CD36 or GLUT) and is taken up into cells via endocytosis.

[0203] In certain embodiments, the bivalent or multivalent agent is MtPTAC. MtPTAC refers to a heterobifunctional molecule consisting of a POI ligand, a mitochondrial caseinolytic protease P (ClpP) ligand, and a linker between these two parts. MtPTAC activates the hydrolase activity of ClpP while simultaneously bringing POI and ClpP into proximity for degradation. In some embodiments, MtPTAC is selected from the group consisting of all compounds, any deuterium-substituted derivatives, analogs, and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier, as described in the publication Dachi Wang et al., Journal of American Chemistry Society, 145(23), 12861-12869, 2023.

[0204] In certain embodiments, the bivalent or multivalent agent is an AUTAC or AUTOTAC. AUTAC is a heterobifunctional molecule composed of a POI ligand, an autophagosome recruitment motif, and a linker between these two moieties. By binding to the POI, the AUTAC molecule can trigger the degradation of the POI by recruiting autophagosomes. The POI can be any suitable protein of interest. AUTOTAC is composed of an autophagy-targeting ligand, a POI ligand, and a linker. AUTOTAC can directly tether the p62 receptor to the POI and induce autophagy of the POI.

[0205] In certain embodiments, AUTAC and AUTOTAC are represented by any of the following compounds: AUTAC1, AUTAC2, AUTAC3, AUTAC4, PHTPP-1304, Vinclozolin M2-2204, Fumagillin-105, PBA-1105, PBA-1106, Anle138b-F105 and PBA-1105b, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0206] In certain embodiments, bivalent or multivalent agents are nucleic acid degradation agents. Examples include but are not limited to ribonuclease targeting chimeras (RIBOTAC), proximity-induced nucleic acid degradation agents (PINAD) or RNA degradation chimeras. RIBOTAC is a heterobifunctional molecule consisting of an RNA targeting ligand, an RNA enzyme recruiting agent or a binding agent and a joint between the two parts. RIBOTAC functions by recruiting endogenous RNA enzymes to specific RNA, activating RNA enzymes and inducing the cracking of target RNA. RNA enzymes include but are not limited to RNA enzyme A, RNA enzyme H, RNA enzyme III, RNA enzyme L, RNA enzyme P, RNA enzyme PhyM, RNA enzyme T1, RNA enzyme T2, RNA enzyme U2, RNA enzyme V, RNA enzyme E, RNA enzyme G, polynucleotide phosphorylase (PNPase), RNA enzyme PH, RNA enzyme R, RNA enzyme D, RNA enzyme T, oligoribonuclease, exoribonuclease I and exoribonuclease II, including all variants, mutations, splice variants, indels and fusions of these listed proteins.

[0207] In certain embodiments, RIBOTAC is represented by any of the following compounds: Compound 2 and Compound 5 in the publication Matthew G. Costales et al., Proceedings of the National Academy of Sciences, 117(5), 2406-2411, 2020, C5-RIBOTAC in the publication Hafeez S. Haniff et al., ACS Central Science, 6(10), 1713-1721, 2020, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0208] In certain embodiments, the bivalent or multivalent agent is a deubiquitinase targeting chimera (DUBTAC), RESTORAC, or ENTAC. DUBTAC, RESTORAC, or ENTAC is a heterobifunctional molecule consisting of a deubiquitinase (DUB) recruiting agent or binder linked to a POI ligand via a linker and used to stabilize the levels of a POI that is degraded in a ubiquitin-dependent manner. The POI can be any suitable protein of interest. DUBs include, but are not limited to, ubiquitin-specific protease (USP / UBP) superfamily proteins such as USP1, USP2, USP3, USP4, USP5, USP6, USP7, USP8, USP9X, USP9Y, USP10, USP11, USP12, USP13, USP14, USP15, USP16, USP17, USP17L2, USP17L3, USP17L4, USP17L5, USP17L7, USP17L8, USP18, USP19, USP20, USP21, USP22, USP23, USP24, USP25, USP26, USP27X, USP28, USP29, USP30, USP31, USP32, USP33, USP34, USP35, USP36, USP37, USP38, USP39, USP40, USP41, USP42, USP43, USP44, USP45, USP46; ovarian tumor (OTU) superfamily proteins, such as OTUB1, OTUB2; Machado-Josephin domain (Machado-Josephin domain) domain, MJD) superfamily proteins, such as ATXN3, ATXN3L; ubiquitin C-terminal hydrolase (UCH) superfamily proteins, such as BAP1, UCHL1, UCHL3, UCHL5; the MINDY family of K48-specific deubiquitinating enzymes, such as MINDY1, MINDY2, MINDY3, MINDY4; and ZUFSP family proteins, such as ZUP, including all variants, mutations, splice variants, insertions, deletions and fusions of these proteins listed.

[0209] In certain embodiments, DUBTAC is represented by any of the following compounds: Publication Jing Liu et al., Journal of American Chemistry Society, 144, 12934–12941, 2022, 05IB9, 11JQ15, 11JQ16, NJH-2-075 (CAS: 2858812-70-9), NJH-2-056 (CAS: 2858812-69-6), NJH-2-057 (CAS: 2858812-70-9), LEB-03-153 (CAS: 2858812-88-9), LEB-03-144 (CAS: 2858812-89-0), LEB-03-145 (CAS: 2858812-90-3), LEB-03-146 (CAS: 2858812-91-4), FOXO-DUBTAC No. 6, p53-DUBTAC No. 6, p53-DUBTAC No. 7 and IRF-DUBTAC No. 7, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0210] In certain embodiments, the bivalent or multivalent agent is a phosphatase recruiting chimera (PhoRC), a dephosphorylation targeting chimera (DEPTAC), or a phosphorylation targeting chimera (PhosTAC). PhoRC, DEPTAC, or PhosTAC is a heterobifunctional molecule consisting of a phosphatase recruiting agent or binding agent connected to a POI ligand via a linker. PhoRC, DEPTAC, or PhosTAC can trigger dephosphorylation of the POI through the function of a protein phosphatase (PP). The POI can be any suitable protein of interest. PPs include, but are not limited to, tyrosine-specific phosphatases, serine / threonine-specific phosphatases, dual-specificity phosphatases, and histidine phosphatases, including all variants, mutations, splice variants, indels, and fusions of these listed target proteins.

[0211] In certain embodiments, PhoRC, DEPTAC or PhosTAC is represented by any of the following compounds: Compound 1, Compound 3, Compound 4a, Compound 5a and Compound 7 in the publication Sayumi Yamazoe et al., Journal of Medicinal Chemistry, 63, 2807-2813, 2020; DDO-3709R8, DDO-3710 and DDO-3711 in the publication Zhang Qiuyue et al., Journal of the American Chemistry Society, 145, 1118-1128, 2023; PhosTAC 7 in the publication Chen PH et al., ACS Chemical Biology, 16, 2808-2815, 2021; any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0212] In certain embodiments, the bivalent or multivalent agent is a regulatory-inducible proximity-targeting chimera (RIPTAC). RIPTAC is a heterobifunctional small molecule that induces a stable ternary complex between a target protein selectively expressed in cancer tissue and a pan-expressed protein essential for cell survival. The resulting synergistic protein-protein interaction (PPI) eliminates the function of the essential protein, thereby selectively leading to the death of cells expressing the target protein.

[0213] In certain embodiments, RIPTAC is represented by any of the following compounds listed in the publication Kanak Raina et al., bioRxiv preprint, 2023, https: / / doi.org / 10.1101 / 2023.01.01.522436, the publication Zonghui Ma et al., Drug Discovery Today, 2023, https: / / doi.org / 10.1016 / j.drudis.2023.103774, and patent WO2023059581A1, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0214] In certain embodiments, the proximity-induced pattern is represented by any compound listed in Chem. Soc. Rev. 52, 5485-5515, 2023, any deuterated derivative, analog, and chelate, or pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and a pharmaceutically acceptable carrier.

[0215] In certain embodiments, the bivalent or multivalent agent is a transcriptional / epigenetic chemical proximity inducer (TCIP). TCIP is a heterobifunctional small molecule that recruits endogenous cancer drivers or downstream transcription factors to the promoters of cell death genes, thereby activating their expression.

[0216] In certain embodiments, TCIP is represented by any compound listed in the publication Sai Gourisankar et al., Nature, 620, 417-425, 2023 and patent WO2022098989A1, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0217] In certain embodiments, the bivalent or multivalent agent is a phosphorylation-inducing chimeric small molecule (PHICS). PHICS are heterobifunctional molecules composed of a kinase activator, a POI binder, and a linker that recruits the kinase to phosphorylate the POI by inducing translocation of substrates, bringing them into proximity. The POI can be any suitable protein of interest. Kinases include, but are not limited to, AMPK and PKC, including all variants, mutations, splice variants, indels, and fusions of these listed target proteins.

[0218] In certain embodiments, PHICS is represented by any of the following compounds: PHICS1, PHICS2, and PHICS3 in the publication Sachini U. Siriwardena et al., Journal of the American Chemistry Society, 142, 14052-14057, 2020, any deuterium-substituted derivatives, analogs, and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0219] In certain embodiments, the bivalent or multivalent agent is an acetylation tag molecule (AceTAG). The AceTAG heterobifunctional molecule consists of a lysine acetyltransferase, a POI binding ligand, and a linker. By binding to the lysine acetyltransferase and the POI, the AceTAG can regulate the distance between the lysine acetyltransferase and the POI, thereby inducing POI acetylation. The POI can be any suitable target protein.

[0220] In certain embodiments, AceTAG is represented by AceTAG-1 in the publication Wesley W. Wang et al., Journal of the American Chemistry Society, 143, 16700-16708, 2021, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0221] In certain embodiments, the bivalent or multivalent agent is a chaperone-mediated protein degrader (CHAMP). The CHAMP heterobifunctional molecule consists of a chaperone-binding ligand, a POI-binding ligand, and a linker. By binding to the chaperone and POI, the CHAMP modulates the distance between the chaperone and POI, thereby inducing POI degradation. The POI can be any suitable protein of interest.

[0222] In certain embodiments, the bivalent or multivalent agent is a BacPROTAC that acts as an endocytosis agent via a bacterial cell membrane protein (such as CD36). The BacPROTAC heterobifunctional molecule consists of a bacterial CIpCP protease binding ligand, a POI binding ligand, and a linker. By binding to the bacterial CIpCP protease and POI, the BacPROTAC can regulate the distance between the CIpCP protease and the POI, thereby inducing POI degradation. The POI can be any suitable protein of interest.

[0223] In certain embodiments, the bivalent or multivalent agent is a caspase cleavage targeting chimera (CACTAC) as an endocytosis agent. The CACTAC heterobifunctional molecule consists of a caspase-binding ligand, a POI-binding ligand, and a linker. By binding to both caspase and POI, CACTAC modulates the distance between caspase and POI, thereby inducing cleavage of the POI. The POI can be any suitable protein of interest.

[0224] In some embodiments, therapeutic agent is a binding agent.Binding agent is any compound combined with membrane or extracellular material, and described membrane or extracellular material include membrane protein or extracellular protein, carbohydrate, lipid, pathogen, particle, virus, bacterium, fungus, protozoan, carrier, cell fragment and another cell.Therefore, described binding agent can be used for internalization cell membrane to form endosome, and subsequently membrane protein, extracellular protein, carbohydrate, lipid, pathogen, particle, virus, bacterium, fungus, protozoan, carrier, cell fragment and another cell are transported to (recipient) cell via endocytosis.Component or extracellular material in internalization membrane, such as protein, carbohydrate, lipid, pathogen, particle, virus, bacterium, fungus, protozoan, carrier, cell fragment and another cell, can finally be degraded by the lysosome of (recipient) cell.

[0225] In some embodiments, therapeutic agent is DNA groove binder or RNA binder.DNA minor groove binder is a crescent molecule, which selectively non-covalently binds to the groove (groove in DNA spiral) of DNA (Sayantan Bhaduri et al., Beilstein Journal of Organic Chemistry, 14,1051-1086,2017).RNA binders are molecules that can be specifically bound to RNA (Jessica L.Childs-Disney et al., Nature Reviews Drug Discovery, 21,736-762,2022). Combination with DNA or RNA with specific sequence usually occurs by directional hydrogen bonding with base pair edges.By being combined with DNA or RNA, the molecule can affect the function of DNA or RNA.

[0226] In some embodiments, the therapeutic agent is a diagnostic agent or a chemical probe. The diagnostic agent or chemical probe comprises a detectable label. Detectable labels include, but are not limited to, binding labels, chromophores, enzyme labels, bioluminescent labels, fluorescent labels, quenchers, radioactive labels, or any other label suitable for detection means. The binding label provides a detectable signal via a binding event. In some embodiments, the binding label can be biotin, a compound used in HaloTag, CLIP-Tag, or SNAP-Tag technology (Jonas Wilhelm et al., Biochemistry, 60(3), 2560-2575, 2021), an antibody, an antigen, or any other label capable of providing a detectable signal via a binding event. The chromophore provides a detectable signal via the absorption and emission of photons. In some embodiments, the chromophore is a fluorophore, a phosphor, a dye, a quantum dot, or any other chromophore capable of absorbing and emitting detectable photons. The enzyme label provides a detectable signal via a reaction with a substrate. The bioluminescent label provides a detectable signal via emission of light from a protein. The quencher or fluorescent label provides a detectable signal via modulation of the photon emission from the chromophore. The radioactive label provides a detectable signal via radioactive decay.As demonstrated in the Examples, conjugated diagnostic agents comprising a fluorophore successfully cross the membrane, but other detectable labels can be incorporated into the cargo.Examples of diagnostic agents or chemical probes include, but are not limited to, Alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680, AMCA, AMCA-S, BODIPY dyes (BODIPY FL, BODIPY ROG, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665), Carboxyrhodamine 6G ( 6G), Carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarins, 4',5-dichloro-2',7'-dimethoxy-fluorescein, DM-NeRF, Eosin, Erythrosine, Fluorescein, FAM, Hydroxycoumarins, IRDye (IRD40, IRD 700, IRD 800), JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue Blue), PyMPO, pyrene, Rhodamine B, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2',4,5',7'-tetrabromosulfone-fluorescein, tetramethylrhodamine (TMR), carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X.

[0227] In certain embodiments, the diagnostic or tracking agent is a multivalent compound containing one or more chemical moieties with fluorescence as a detectable label. The emission and excitation wavelengths of the diagnostic agent are, but are not limited to, 200 nm to 900 nm. The diagnostic or tracking agent can be used for in vitro and in vivo imaging.

[0228] In certain embodiments, the endocytic agent is an exocytic vesicle (or an endocytic agent-vesicle complex). An exocytic vesicle refers to a vesicle or vesicle composition comprising a vesicle-forming component or particle and one or more endocytic agents associated with the vesicle-forming component or particle, wherein the vesicle-forming component includes any cytoplasm of the cell and the membrane component mediated by endocytosis, including nucleic acids, proteins, lipids, carbohydrates and metabolites. The endocytic agent may be located at any site in the vesicle, including encapsulation or embedding in the vesicle-forming component formed by lipids, attachment to the membrane component (inward and / or outward), insertion or penetration of the membrane component. The endocytic agent may bind to the vesicle via a covalent or non-covalent bond with a binding affinity K of less than 20.0 mM. D Binds to any component of the vesicle.

[0229] The diameter of the exocytic vesicle is less than 10,000 nm. In certain embodiments, the diameter of the exocytic vesicle is 10 to 10,000 nm. In certain embodiments, the diameter of the exocytic vesicle is 10 to 1000 nm.

[0230] In certain embodiments, the exocytic vesicle can be an extracellular vesicle (EV) containing an endocytic agent or associated with an endocytic agent in any equivalent amount. EVs include, but are not limited to, exosomes, microvesicles, microparticles, apoptotic bodies, oncosomes, extranuclear granules, synaptic vesicles, prostatic bodies, and matrix vesicles. The endocytic agent can be selected from any endocytic agent as defined in the present disclosure. In the exocytic vesicle, the endocytic agent can be present at any site of the EV and / or can be bound to any component of the EV via covalent or non-covalent bonds.

[0231] In certain embodiments, exocytic vesicles can be generated by any cell and can be secreted from the cell to the extracellular space by exocytosis, endocytic transport and / or membrane fusion. In certain embodiments, exocytic vesicles can be generated in vitro and / or in vivo by mixing vesicle-forming components or particles (including EVs) with endocytic agents in any equivalent amount in any suitable solution, matrix or body fluid, wherein the endocytic agent can form a covalent or non-covalent bond with the vesicle-forming components or particles (including EVs).

[0232] Based on the fact that foreign substances can be taken up by cells and released from (donor) cells in the form of extracellular vesicles (EVs), and that extracellular vesicles can be taken up by any of the same cells or different cells via exocytosis or membrane fusion (Ravi Shah et al., The New England Journal of Medicine, 8 (379), 958-966, 2018; Oscar PB Wiklander et al., Science Translational Medicine, 11 (492), eaav8521, 2019; Raghu Kalluri et al., Science, 367 (6478), eaau6977, 2020), the present exocytic vesicles can be used to transport any endocytic agent as defined in the present disclosure into cells. In addition, the present exocytic vesicles can be used to cross membrane barriers (including the blood-retinal barrier, the lung endothelial and epithelial barriers, the skin barrier, and the brain-blood barrier), and / or deliver any endocytic agent as defined in the present disclosure to any target tissue and organ in an animal body. In particular, the present exocytic vesicles are suitable for the delivery of endocytic agents for the research, diagnosis, prevention, and treatment of ocular, respiratory, skin, and CNS conditions and diseases.

[0233] In this disclosure, an exocytic vesicle is referred to as an endocytic agent. All benefits and methods associated with endocytic agents are applicable to exocytic vesicles.

[0234] Membrane components that mediate endocytosis

[0235] Membrane components that mediate endocytosis include proteins, lipids, and carbohydrates that are components of membranes, including cell membranes, that mediate the process of endocytosis or whose binding allows for increased uptake via endocytosis.

[0236] In some embodiments, lipids include glycolipids, phospholipids, ceramides, and cholesterol.

[0237] In some embodiments, the carbohydrate is a monosaccharide or a molecule composed of 2-100 monosaccharide units and / or derivatives, and can be linear or branched, attached to a protein to form a glycoprotein, or to a lipid to form a glycolipid.

[0238] In certain embodiments, carbohydrates include, but are not limited to, N-acetylgalactosamine (GalNAc), chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), hyaluronic acid, and sialic acid (SA).

[0239] In some embodiments, proteins include, but are not limited to, integral membrane proteins, peripheral membrane proteins, lipid-anchored proteins, globular proteins, and glycoproteins, including all variants, mutations, splice variants, substitutions, and fusions.

[0240] In certain embodiments, proteins include, but are not limited to, ATP-binding cassette (ABC) transporters (such as MDR1 / 2 / 3 / 4 / 5 and ABCG2), anaplastic lymphoma kinase (ALK), anamniotic fluid, AMPA receptor (AMPAR) (Maria Fiuza et al., Journal of Cell Biology, 216(10), 3323-3338, 2017), apolipoprotein E receptor (ApoER), amino acid transporters, amyloid precursor protein (APP), ileal apical sodium / bile acid cotransporter (ASBT), alanine serine cysteine ​​transporter (ASCT), asialoglycoprotein receptor (ASPGPR), V-type proton ATPase 6 (ATP6V), ATP6V1H, avidin, beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), beta2-adrenergic receptor, CDO brothers (Boc), brassinosteroid insensitive-1 (BRI 1), cell adhesion molecule (CAM) receptors, coxsackievirus-adenovirus receptor (CAR), chemokine receptors (such as CC chemokine receptor (CCR) and CXC chemokine receptor (CXCR), such as CXCR2, CXCR4 and CXCR7), cluster of differentiation (CD) (such as CD4, CD9, CD13 / APN receptor, CD25, CD26 (DPP4), CD30, CD33, CD36, CD38, CD46, CD56, CD63, CD71, CD74, CD80, CD81, CD82, CD86, CD123, CD138, CD147, CD152, CD152 (CTLA4), CD166, CD174, CD197, CD205, CD227, CD228, CD269, CD276, and CD326), cation-dependent MPR (CD-MPR), Cdon, cystic fibrosis transmembrane conductance regulator (CFTR), cation-independent MPR / insulin-like growth factor-II (IGF-II) receptor (CI-MPR), IGF-2R, chemokine receptors, human collectin placenta 1 (CL-P1), cone opsins, connexins, cell-penetrating peptides (CPP), C SF2R, CSF1R / FM, excitatory amino acid carrier 1 (EAAC1), E-cadherin, early endosomal autoantigen 1 (EEA1), epithelial cell adhesion molecule (EpCAM), erythropoietin-producing hepatocellular carcinoma receptor (EphR), endothelial cell protein C receptor (EPCR), fatty acid binding protein (FABP), fatty acid transport protein (FATP), major facilitator superfamily domain protein 2 (mfsd2), neonatal Fc receptor (FCRN), free fatty acid receptor (FFAR), vesicoplanin-1, vesicoplanin-2, folate receptors (such as reduced folate carrier,FOLT1 / 2 / 3 and proton-coupled folate transporter (PCFT), Frizzled4, gamma-aminobutyric acid type A receptor (GABAAR), growth arrest-specific 1 (Gas1), glutamate transporter (GLT), glucose transporter (GLUT), glutathione transporter, ionotropic AMPA glutamate receptor (GluR), GLP1, glycoproteins (such as gp18, gp31 and gp60), glucokinase, G protein-coupled receptors (GPCRs) (such as GPR20, GPR20, free fatty acid receptor 1 (GPR40), GPR119 and GPR120), HDLR, IFN-γR, intermediate density lipoprotein receptor (IDLR), IL-10R, IL-4R, integrins (such as αV β3, α4β1 and α5β1 integrins), insulin receptor (IR), insulin-like growth factor receptor, potassium-chloride cotransporter 2 (KCC2), inwardly rectifier potassium channel (Kir2.3), lactoferrin receptor, L-amino acid transporter (LAT), lysosomal associated membrane protein (LAMP), low-density lipoprotein receptor (LDLR), low-density lipoprotein receptor-related protein (LRP), major facilitator superfamily domain protein 2 (Mfsd2a), monocarboxylate transporter (MCT), multidrug resistance protein (MRP), MET, metabotropic glutamate receptor (mGlu1), major histocompatibility complex class II molecule (MHC-II), MHC-I, MINCLE, multidrug resistance protein (MRP), N-cadherin, K, + Dependent Na + / Ca 2+exchanger 2 (NCKX2), nicotinic acetylcholine receptor, NOTCH receptor, Niemann-Pick C1-like protein 1 (NPC1L1), N-methyl D-aspartate receptor (NR), nucleoside transporter (NT), sodium / taurocholate co-transporting peptide (NTCP), organic anion transporter (OAT), organic anion transporting polypeptide (OATP), organic cation transporter (OCT), organic solute transporter (OST), otoferlin, P2X purinergic receptor 4 (P2x4R), purinergic receptor P2Y12 (P2Y12), protease-activated receptor (PAR), PEIZO1, PEIZO2, peptide transporter (PEPT), p-glycoprotein (P-gp), peroxisome proliferator-activated receptor (PPAR), prostate-specific membrane antigen (PSMA), receptor patched 1 (PTCH1), RAS, retinol binding protein (RBP), riboflavin transporter protein (RFVT), rhodopsin, ribonuclease K (RNASEK), RING finger protein (RNF), renal outer medullary potassium channel (ROMK), receptor serine / threonine kinases (RSKs) (such as transforming growth factor beta (TGF-β) receptor and proteins listed in Aristidis Moustakas et al., Receptor Serine / Threonine Kinases (ISBN: 978-3-540-44244-8)), receptor tyrosine kinases (RTKs) (such as fibroblast growth factor receptor (FGFR), FLT1 / 2 / 3 / 4, FGFR1 / 2 / 3 / 4, RET, tropomyosin-related kinase (TRK), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER), vascular endothelial growth factor receptor (VEGFR), TYRO3, and as described in the publication Mark A.Lemmon and Joseph Schlessinger, Cell, 141(7)1117-1134, 2010), Sanpodo / Notch, solute carrier (SLC) transporters (such as hMATE1 and proteins listed in the publication Enrico Girardi et al., Nature Chemical Biology, 16, 469-478, 2020), solute carrier family 37 member A3 (SLC37A3), SGLT, sodium-coupled monocarboxylate transporter (SMCT), sodium-dependent multivitamin transporter (SMVT), synaptosome-associated protein (SNAP), sortilin-related CNS expressed 1a (SorCS1a), SorCS1c, secretory protein acidic and rich in cysteine ​​(SPARC), SPv-NK1R, SRC, scavenger receptors (SR) (such as CD36, LAMP1 and LAMP2), syntaxin (STX), sodium-vitamin C cotransporter (SVCT), synaptotagmin thyroxine (SYT), triiodothyronine (T3), transferrin receptor (TfR), transforming growth factor β (TGF-β), trans-Golgi network (TGN38), thiamine transporter (THTR), toll-like receptor (TLR) (such as TLR4), TPN1p, vesicle-associated membrane protein (VAMP), vitamin D receptor (VDR), vesicular GABA transporter (VGAT), vesicular glutamate transporter (VGLUT), very low-density lipoprotein receptor (VLDLR), urate transporter 1 (URAT1) and zinc ring finger protein (ZRNF), including all variants, mutations, splice variants, indels and fusions of these target proteins listed. Similar transporters or receptors expressed on microbial cell membranes are also suitable for such BacPROTACs.

[0241] Chemistry and Terminology

[0242] As used herein,

[0243] indicates the binding site;

[0244] Is a single bond or a double bond;

[0245] R, R' and R" are each independently H, D, O, =O, S, =S, SS, =NH, =N(OH), N(OH), N(H)O, CH2F, CHF2, CF3, 1-100 polyethylene glycol, C1-C100 alkoxy, C1-C100 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkylene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or C1-C100 heteroaryl, 1-100 polyethylene glycol-R a 、C1-C100 alkyl-R a 、C2-C100 alkylene-R a 、C2-C100 alkyne-R a 、C3-C100 cycloalkyl-R a 、C3-C100 cycloalkylene-R a 、C3-C100 Cycloalkyne-R a 、C3-C100 heterocyclyl-R a 、C6-C100 aryl-R a or C1-C100 heteroaryl-R a 、B(OR a 2) Si(OR a 3) C≡C, C≡CR a 、CH=CH2、CH=CHR a 、CH=C(R a 2. CR a =CHR a , CR a =CR a , C(O), CR a =C(R a )2, COR a ,CONH2,C(O)OR a ,OC(O)R a ,OC(O)OR a , OC(O)N(R a )2, CONHR a ,CON(R a )2,NH2,N(H)R a ,N(R a )2,N + (R a )3,NHNH2,NHN(H)R a ,-NHN(R a )2,NHC(O)R a ,NHC(O)OR a ,NHC(O)NH2,NHC(O)NHR a,NHC(O)N(R a )2,NR a C(O)NH2,NR a C(O)NHR a ,NR a C(O)N(R a )2,NR a C(S)N(R a )2,NHC(NH)NH2,NHC(NH)NHR a ,NHC(NH)N(R a )2,NR a C(NH)NH2,NR a C(NH)NHR a ,NR a C(NH)N(R a )2,NR a C(NR a )N(R a )2, NHS(O)R a ,NHS(O)NH2,NHS(O)NHR a ,NHS(O)N(R a )2,NR a S(O)NH2,NR a S(O)NHR a ,NR a S(O)N(R a )2,NHS(O)2R a ,NHS(O)2NH2,NHS(O)2N(H)R a ,NHS(O)2N(R a )2,NR a S(O)2NH2,NR a S(O)2NHR a ,NR a S(O)2N(R a )2, OH, OR a ,OS(O)2OH,OS(O)2OR a ,OS(O)OH,OS(O)OR a ,OS(O)2NH2,OS(O)2N(H)R a ,OS(O)2N(R a )2, OS(O)NH2, OS(O)N(H)R a ,OS(O)N(R a )2, SH, SR a ,S(O)R a ,S(O)2NH2,S(O)2NHR a,S(O)2N(R a )2,S(O)N(H)R a ,S(O)N(R a )2, SON(R a )3, SO2H, SO2R a , P(O)(OH)2, P(O)(OH)(OR a ), P(O)(OR a )2,P(O)(OH)OP(O)(OH)2,P(O)(OH)OP(O)(OH)(OR a ), P(O)(OH)OP(O)(OR a )2,P(O)(OH)OP(O)(OH)OP(O)(OH)2,P(O)(OH)OP(O)(OH)OP(O)(OH)(OR 3 ), P(O)(OH)OP(O)(OH)OP(O)(OR a )2,OP(O)(OR a ), OP(O)OP(O)(OH)2, OP(O)OP(O)(OH)(OR a ), OP(O)OP(O)(OR a )2, OP(O)OP(O)(OH)OP(O)(OH)2, OP(O)OP(O)(OH)OP(O)(OH)(OR a ), OP(O)OP(O)(OH)OP(O)(OR a )2,

[0246]

[0247]

[0248]

[0249] MBCF, SMDA, LA, GA, PPA, VtA, AAP, SCA, NNNA, RMPB, ADD, an agent or a portion of an agent, any deuterium-substituted derivative, or any combination thereof, wherein the interrupting group and one or both capping groups can be the same or different;

[0250] U, V, U', V', V", V"', W, W', X', R a 、R a 、R b 、R b 、R c 、R c 、R d 、R d 、Re 、R f 、R g and R h Independently selected from -H, -D, -F, -Cl, - 18 F, -CH3, -CF3, -CDH2, -CD2H, -CD3F, Cl, Br, CF3, CHF2, CH2F, OH, SH, NH2, NHNH2, COOH, CONH2, SONH2, SO2NH2, B(OH2), Si(OH3), NHC(NH)NH 2、 C(O)(NH)OH, P(O)(OH)2, P(O)(OH)OP(O)(OH)2, P(O)(OH)OP(O)(OH)OP(O)(OH)2, R i F, R i Cl, R i Br, R i CF3, R i CHF2, R i CH2F、R i OH, R i SH, R i NH2, R i NHNH2、R i COOH、C(O)OR i 、OC(O)R i 、R i CONH2, R i SONH2, R i SO2NH2、R i B(OH)2、R i Si(OH)3、R i NHC(NH)NH2、R i C(O)(NH)OH、R i P(O)(OH)2、R i P(O)(OH)OP(O)(OH)2、R i P(O)(OH)OP(O)(OH)OP(O)(OH)2, an agent or a portion of an agent, any deuterium-substituted derivative, or any combination thereof, wherein the interrupting group and one or both of the capping groups may be the same or different, wherein R a Available in 1-100 polyethylene glycol-R a 、C1-C100 alkyl-R a 、C2-C100 alkylene-R a 、C2-C100 alkyne-R a 、C3-C100 cycloalkyl-R a 、C3-C100 cycloalkylene-R a、C3-C100 Cycloalkyne-R a 、C3-C100 heterocyclyl-R a 、C6-C100 aryl-R a or C1-C100 heteroaryl-R a any position of the 1-100 polyethylene glycol, C1-C100 alkoxy, C1-C100 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkylene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or C1-C100 heteroaryl group;

[0251] R i is 1-100 polyethylene glycols, C1-C100 alkoxy, C1-C100 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkylene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl or C1-C100 heteroaryl, a linker, any deuterium-substituted derivative or any combination thereof, wherein the interrupting group and one or two capping groups may be the same or different.

[0252] Chemical bond or linker unit Can be combined with chemical bonds or linker units Share the same structure and bind to any site of a ligand, E3 ligase ligand, agent, chemical arm, SMDA, LA, Ga, PPA, VtA, AAP, SCA, NNNA, RMPB, or ADD;

[0253] m, n and q are independently selected from 0 to 100, including any number therebetween.

[0254] The term "substituted" means that the specified group or moiety bears one or more suitable substituents, wherein the substituents may be attached to the specified group or moiety at one or more positions. For example, an aryl group substituted with a cycloalkyl group may indicate that the cycloalkyl group is attached to an atom of the aryl group by a bond, or by being fused to the aryl group and sharing two or more common atoms.

[0255] Unless otherwise specifically defined, "aryl" means a cyclic aromatic hydrocarbon group having 1 to 100 aromatic rings, including monocyclic or bicyclic groups such as phenyl, biphenyl or naphthyl. When containing two aromatic rings (e.g., bicyclic), the aromatic rings of the aryl group are optionally joined at a single point (e.g., biphenyl) or fused (e.g., naphthyl). The aryl group is optionally substituted with one or more substituents (e.g., 1 to 100 substituents) at any point of attachment. Exemplary substituents include, but are not limited to, -H, -halogen, -CN, -O(C1-C100)alkyl, -(C1-C100)alkyl, -O(C2-C100)alkenyl, -O(C2-C100)alkynyl, -(C2-C100)alkenyl, -(C2-C100)alkynyl, -OH, -OP(O)(OH)2, -OC(O)(C1-C100)alkyl, -C(O)(C1-C100)alkyl, -OC(O)O(C1-C100)alkyl, -NH2, -NH((C1-C100)alkyl), -N(C1-C100)alkyl)2, -S(O)2-, -(C1-C100)alkyl, -S(O)NH(C1-C100)alkyl, and -S(O)N((C1-C100)alkyl)2. The substituent itself is optionally substituted. In addition, when containing two fused rings, the aryl group optionally has an unsaturated or partially saturated ring fused to a fully saturated ring. Exemplary ring systems of these aryl groups include but are not limited to phenyl, biphenyl, naphthyl, anthracenyl, phenalenyl, phenanthrenyl, indanyl, indenyl, tetrahydronaphthyl, tetrahydrobenzoannulyl, etc.

[0256] Unless expressly defined otherwise, "heteroaryl" means a monovalent monocyclic aromatic group or polycyclic aromatic group having 3 to 100 ring atoms, which contains one or more ring heteroatoms selected from N, O or S, with the remaining ring atoms being C. Heteroaryl as defined herein also refers to bicyclic heteroaromatic groups, wherein the heteroatoms are selected from N, O or S. Aromatic groups are optionally substituted independently with one or more substituents as described herein. Examples include, but are not limited to, furanyl, thienyl, pyrrolyl, pyridinyl, pyrazolyl, pyrimidinyl, imidazolyl, isothiocyanate, thiophene ... Azolyl, Azolyl, oxadiazolyl, pyrazinyl, indolyl, thiophen-2-yl, quinolyl, benzopyranyl, isothiazolyl, thiazolyl, thiadiazole, indazole, benzimidazolyl, thieno[3,2-b]thiophene, triazolyl, triazinyl, imidazo[1,2-b]pyrazolyl, fluoro[2,3-c]pyridinyl, imidazo[1,2-a]pyridinyl, indazolyl, pyrrolo[2,3-c]pyridinyl, pyrrolo[3,2-c] pyridyl, pyrazolo[3,4-c]pyridyl, thieno[3,2-c]pyridyl, thieno[2,3-c]pyridyl, thieno[2,3-b]pyridyl, benzothiazolyl, indolyl, indolinyl, indolinone, dihydrobenzothienyl, dihydrobenzofuranyl, benzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, dihydrobenzooxanyl, quinolinyl, Isoquinolyl, 1,6-naphthyridinyl, benzo[de]isoquinolyl, pyrido[4,3-b][1,6]naphthyridinyl, thieno[2,3-b]pyrazinyl, quinazolinyl, tetrazolo[1,5-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl, isoindolyl, pyrrolo[2,3-b]pyridinyl, pyrrolo[3,4-b]pyridinyl, pyrrolo[3,2-b] Pyridyl, imidazo[5,4-b]pyridyl, pyrrolo[1,2-a]pyrimidinyl, tetrahydropyrrolo[1,2-a]pyrimidinyl, 3,4-dihydro-2H-1Δ2-pyrrolo[2,1-b]pyrimidine, dibenzo[b,d]thiophene, pyridin-2-one, fluoro[3,2-c]pyridyl, fluoro[2,3-c]pyridyl, 1H-pyrido[3,4-b][1,4]thiazinyl, benzo[ Azolyl, benzyl oxazolyl, fluoro[2,3-b]pyridinyl, benzothiophenyl, 1,5-naphthyridinyl, fluoro[3,2-b]pyridine, [1,2,4]triazolo[1,5-a]pyridinyl, benzo[1,2,3]triazolyl, imidazo[1,2-a]pyrimidinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazole, 1,3-dihydro-2H-benzo[d]imidazol-2-one, 3,4-dihydro-2H-pyrazolo[1,5-b][1,2] [1,5-a]pyridinyl, thiazolyl, imidazo[2,1-b][1,3,4]thiadiazolyl, thieno[2,3-b]pyrrolyl, 3H-indolyl and derivatives thereof. In addition, when containing two fused rings, the aryl groups defined herein may have an unsaturated or partially saturated ring fused to a fully saturated ring. Exemplary ring systems of these heteroaryl groups include indolinyl, indolinonyl, dihydrobenzothiophenyl, dihydrobenzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, 3,4-dihydro-1H-isoquinolinyl, 2,3-dihydrobenzofuran, indolinyl, indolyl and dihydrobenzoxanyl.

[0257] Halogen or "halo" refers to fluorine, chlorine, bromine, iodine and their isotopic isomers.

[0258] "Alkyl" means a straight or branched chain saturated hydrocarbon containing 1 to 100 carbon atoms. Examples of (C1-C100)alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and isohexyl.

[0259] "Alkoxy" means a straight or branched saturated hydrocarbon containing 1-100 carbon atoms with a terminal "O" in the chain (e.g., O(alkyl)). Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, tert-butoxy, or pentoxy.

[0260] "Alkenyl" means a straight or branched chain unsaturated hydrocarbon containing 2-100 carbon atoms. An "alkene" contains at least one double bond in the chain. The double bond of an alkenyl group can be unconjugated or conjugated with another unsaturated group. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, pentenyl, or hexenyl. An alkenyl group can be unsubstituted or substituted and can be straight or branched.

[0261] "Alkynyl" means a straight or branched chain unsaturated hydrocarbon containing 2-100 carbon atoms. An "alkyne" group contains at least one triple bond in the chain. Examples of alkynyl groups include ethynyl, propargyl, n-butynyl, isobutynyl, pentynyl, or hexynyl. Alkynyl groups can be unsubstituted or substituted.

[0262] "Cycloalkyl" or "carbocyclyl" means a monocyclic or polycyclic saturated carbocyclic ring containing 3-100 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, norbornenyl, bicyclo[2.2.2]octyl or bicyclo[2.2.2]octenyl, and derivatives thereof. (C3-C100)cycloalkyl groups are cycloalkyl groups containing 3 to 100 carbon atoms. Cycloalkyl groups can be fused (e.g., decalin) or bridged (e.g., norbornane).

[0263] "Cycloalkylene" means a monocyclic or polycyclic carbocyclic ring containing 3-100 carbon atoms. "Cycloalkylene" contains at least one double bond in the chain. The double bond of a cycloalkylene group can be unconjugated or conjugated with another unsaturated group. Examples of cycloalkylene groups include cyclopropenyl, cyclobutenyl, butadienyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, cycloheptenyl, cycloheptadienyl, cycloheptatrienyl, cyclooctenyl, cyclooctadienyl, or cyclooctatetraenyl.

[0264] "Cycloalkyne" means a monocyclic or polycyclic carbocyclic ring containing 3-100 carbon atoms. A "cycloalkyne" group contains at least one triple bond in the chain. The triple bond of a cycloalkyne can be unconjugated or conjugated with another unsaturated group. Examples of cycloalkyne groups include cyclooctynyl.

[0265] "Heterocyclyl" or "heterocycloalkyl" means a saturated or partially saturated monocyclic or polycyclic ring containing carbon and at least one heteroatom selected from oxygen, nitrogen, or sulfur (O, N, or S), and in which there are no shared delocalized pi electrons between the ring carbons or heteroatoms (aromaticity). The heterocycloalkyl ring structure may be substituted with one or more substituents. The substituents themselves may be optionally substituted. Examples of heterocyclyl rings include, but are not limited to, oxetanyl, azetadinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, Oxazoline, oxazolidinyl, thiazolinyl, thiazolidinyl, pyranyl, thiopyranyl, tetrahydropyranyl, dioxolanyl, piperidinyl, morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S-dioxide, piperazinyl, azepine, oxepinyl, diazepine, tropane, Oxazolidinone, 1,4-dioxadiazol alkyl, dihydrofuranyl, 1,3-dioxolanyl, imidazolidinyl, imidazolinyl, dithiolanyl and homotropyl.

[0266] "Haloalkyl" means an alkyl group substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, or trichloromethyl.

[0267] "Haloalkoxy" means an alkoxy group substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethoxy, difluoromethoxy, pentafluoroethoxy, or trichloromethoxy.

[0268] "Cyano" means a substituent having a carbon atom triple-bonded to a nitrogen atom, for example, C≡N.

[0269] "Amino" means a substituent containing at least one nitrogen atom (eg, -NH2).

[0270] "Isomers" means compounds that have the same number and kind of atoms, and therefore the same molecular weight, but differ with regard to the arrangement or configuration of the atoms in space. The term includes stereoisomers and geometric isomers.

[0271] "Stereoisomer" or "optical isomer" means a stable isomer that has at least one chiral atom or restricted rotation to produce a perpendicular asymmetric plane (e.g., certain biphenyls, allenes, and spirocyclic compounds) and can rotate the plane of plane-polarized light. Since asymmetric centers and other chemical structures are present in the compounds of the present disclosure that can produce stereoisomerism, the present disclosure contemplates stereoisomers and mixtures thereof. The compounds of the present disclosure and their salts include asymmetric carbon atoms and can therefore exist as single stereoisomers, racemates, and as mixtures of enantiomers and diastereomers. Typically, such compounds will be prepared as racemic mixtures. However, if desired, such compounds can be prepared or separated into pure stereoisomers, i.e., as single enantiomers or diastereomers, or as stereoisomer-enriched mixtures. As discussed in more detail below, individual stereoisomers of a compound are prepared by synthesis from optically active starting materials containing the desired chiral center, or by preparing a mixture of enantiomeric products followed by separation or resolution (such as conversion to a mixture of diastereomers followed by separation or recrystallization, chromatography techniques, use of a chiral resolving agent, or direct separation of enantiomers on a chiral chromatographic column). Starting compounds with specific stereochemistry are either commercially available or prepared by the methods described below and resolved by techniques well known in the art.

[0272] "Enantiomers" means a pair of stereoisomers that are non-superimposable mirror images of each other.

[0273] "Diastereomers" or "diastereomers" means optical isomers that are not mirror images of one another.

[0274] "Racemic mixture" or "racemate" means a mixture containing equal parts of individual enantiomers.

[0275] "Non-racemic mixture" means a mixture containing unequal parts of individual enantiomers.

[0276] "Geometric isomers" means stable isomers resulting from restricted rotational freedom around double bonds (e.g., cis-2-butene and trans-2-butene) or in cyclic structures (e.g., cis-1,3-dichlorocyclobutane and trans-1,3-dichlorocyclobutane). Since carbon-carbon double (olefinic) bonds, C=N double bonds, cyclic structures, etc. can be present in the compounds of the present disclosure, the present disclosure contemplates each of the various stable geometric isomers and mixtures thereof resulting from the arrangement of substituents around these double bonds and in these cyclic structures. Substituents and isomers are designated using the cis / trans convention or using the E or Z system, where the term "E" means that higher-order substituents are on opposite sides of the double bond, and the term "Z" means that higher-order substituents are on the same side of the double bond. A thorough discussion of E and Z isomerism is provided in J. March, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 4th ed., John Wiley & Sons, 1992, which is incorporated herein by reference in its entirety.

[0277] Some compounds of the present disclosure may exist in more than one tautomeric form. As stated above, the compounds of the present disclosure include all such tautomers.

[0278] It is well known in the art that the biological and pharmacological activities of compounds are sensitive to the stereochemistry of the compound. Thus, for example, enantiomers often exhibit significantly different biological activities, including differences in pharmacokinetic properties, metabolism, solubility, protein binding, etc., as well as pharmacological properties, including differences in the type of activity exhibited, the degree of activity, toxicity, etc. Therefore, those skilled in the art will understand that one enantiomer may be more active or may exhibit beneficial effects when enriched relative to the other enantiomer or when separated from the other enantiomer. In addition, those skilled in the art will know how to separate, enrich, or selectively prepare the enantiomers of the disclosed compounds based on the present disclosure and the knowledge of the prior art.

[0279] Thus, although the racemic form of a drug can be used, it is often not as effective as administering an equal amount of an enantiomerically pure drug. Thus, if one enantiomer is pharmacologically more active, less toxic, or has a preferred disposition in the body than the other enantiomer, it would be therapeutically beneficial to preferentially administer that enantiomer.

[0280] The preparation of pure enantiomers or mixtures having the desired enantiomeric excess (ee) or enantiomeric purity can be accomplished by one or more of a number of methods known to those skilled in the art for (a) separation or resolution of enantiomers, or (b) enantioselective synthesis, or by a combination of these methods.

[0281] These resolution methods often rely on chiral recognition, and such methods are generally disclosed in Chiral Separation Techniques: A Practical Approach (2nd ed.), G. Subramanian (ed.), Wiley-VCH, 2000; TE Beasley and RP W Scott, Chiral Chromatography, John Wiley & Sons, 1999; and Satinder Ahuja, Chiral Separations by Chromatography, Am. Chem. Soc., 2000.

[0282] In general, all tautomeric and isomeric forms and mixtures of a chemical structure or compound, whether individual geometric isomers or stereoisomers or racemic or non-racemic mixtures, are contemplated unless a specific stereochemistry or isomeric form is explicitly indicated in the compound name or structure.

[0283] "Charged" refers to the ionic form of an atom or group of atoms in which the number of electrons is not equal to the number of protons. A charged atom or group of atoms can be anionic (negatively charged or negatively charged) and cationic (positively charged or positively charged) at certain pH values. "Chargeable" or "chemically chargeable" refers to the ability of an atom or group of atoms to be ionized in aqueous solution at certain pH values.

[0284] "A chemical bond or moiety that can form a reversible or irreversible covalent bond with any nucleophile in biology" refers to any chemical bond or moiety that can reversibly or irreversibly cross-link an endocytosis agent and any nucleophile in biology (including lysine, arginine, cysteine, serine, threonine, and carbohydrates) via a covalent bond. In particular, "a chemical bond or moiety that can form a reversible covalent bond with any nucleophile in biology" refers to any chemical bond or moiety that can cross-link an endocytosis agent and any nucleophile in biology via a covalent bond, but the dissociation of the covalent bond can be broken and / or reformed under any conditions. In certain embodiments, "a chemical bond or moiety that can form a reversible or irreversible covalent bond with any nucleophile in biology" can be selected from any chemical bond, linkage or moiety listed in patents WO2020252397A1 and WO2011018611A1 and publications Anupam Bandyopadhyay et al., Current Opinion Chemical Biology, 34, 110-116, 2016; Hannah Kiely-Collins et al., Cell Chemical Biology, 28(7), 952-968, 2021; Fandi Sutanto et al., RSC Medicinal Chemistry, 11, 876-884, 2020 and Sijie Wang et al., ChemRxiv preprint, 2022 (DOI: 10.26434 / chemrxiv-2022-tvgn1). In certain embodiments, “a chemical bond or moiety that can form a reversible or irreversible covalent bond with any nucleophile in biology” includes, but is not limited to, substituted or unsubstituted Michael acceptors (including acrylamide, acrylate), cyclic imine, thiol, α-cyanoacrylamide (or acrylate), α-substituted acrylonitrile, α, β-diketoamide with arginine (Ziyang Zhang et al., Journal of the American Chemistry Society, 144(35), 15916-15921, 2022), β-lactone derivatives (Ziyang Zhang et al., Nature Chemical Biology, 18, 1177-1183, 2022), benzylidine rhodanine ... derivative), a disulfide bond, a boronic acid, an α-ketoamide, a nitrile moiety, a methyl ester, a ketone, an o-phenoxy moiety, an aromatic aldehyde, coumarin 3-aldehyde, an o-boronic acid substituted benzaldehyde (or acetophenone) and any deuterium substituted derivative, or any combination thereof, wherein the interrupting group and one or both of the capping groups may be the same or different.

[0285] Methods and drug administration

[0286] One aspect of the present technology provides methods of affecting cellular uptake of an agent via endocytosis.

[0287] In some embodiments, the method refers to the use of structural modification strategies used in the art to allow or adjust the binding affinity and / or binding valence of the agent to the membrane component that mediates endocytosis, dimerization or clustering of the membrane component that mediates endocytosis, and / or conformational changes of the membrane component that mediates endocytosis to enhance endocytic efficacy and / or efficiency. Structural modification strategies include, but are not limited to, adding or removing substituents, fragment replacement, cyclization, backbone transitions, bioisostericity, linkerology (linker-activity relationship studies), and prodrugs. This method allows for enhancing the expected biological activity of the agent and the binding affinity of the agent to the membrane component that mediates endocytosis to enhance the endocytic efficacy and / or efficiency for disease diagnosis and treatment. In some embodiments, the method refers to covalently conjugating a chemical arm to the agent via a cleavable or non-cleavable chemical bond or linker to generate a multivalent endocytic agent. Multivalent endocytic agents can have better absorption via enhanced endocytosis for disease diagnosis and treatment. In certain embodiments, the method refers to covalently conjugating a chemical arm to an agent via a chemical bond or linker to generate a multivalent endocytosis agent, wherein the chemical arm is selected to allow or increase the binding affinity and / or binding valence of the resulting multivalent endocytosis agent to a membrane component that mediates endocytosis, dimerization or aggregation of the membrane component that mediates endocytosis, and / or conformational changes of the membrane component that mediates endocytosis, to enhance endocytosis efficacy and / or efficiency without sacrificing the intrinsic pharmacological activity of the agent. In certain embodiments, the method refers to covalently conjugating a chemical arm to a therapeutic agent via a cleavable bond or linker, wherein the chemical arm is selected to weaken, reduce, or remove the intended function of the therapeutic agent until the chemical arm is cleaved from the therapeutic agent. This method allows for enhanced targeted delivery to cells or tissues via endocytosis.

[0288] In some embodiments, the method refers to the use of a structural modification strategy for allowing or enhancing conformational changes in membrane components that mediate endocytosis by structurally modifying the endocytosis agent for any purpose, wherein the structural modification strategy includes but is not limited to preparing charged molecules or salt-forming techniques in the art, forming multivalent endocytosis agents by connecting charged, chargeable, and other hydrophilic chemical arms, or using medicinal chemistry strategies in the art (including the introduction of reversible or irreversible covalent bonds or moieties) for structural modification. In certain embodiments, a method for allowing or enhancing conformational changes in membrane components that mediate endocytosis to enhance the endocytic efficiency and / or efficacy of the endocytosis agent is to increase the binding affinity and / or binding valence of the endocytosis agent to the membrane component that mediates endocytosis. In certain embodiments, a method for allowing or enhancing conformational changes in membrane components that mediate endocytosis to enhance the endocytic efficiency and / or efficacy of the endocytosis agent is to increase the dimerization or clustering of membrane components that mediate endocytosis. In certain embodiments, a method for allowing or enhancing conformational changes in membrane components that mediate endocytosis to enhance the endocytic efficiency and / or efficacy of endocytic agents is to adjust the environmental factors of membrane components that mediate endocytosis, the interaction with membrane cofactor proteins and / or the post-translational modification status of membrane components that mediate endocytosis and membrane cofactor proteins by utilizing structural modification strategies used in the field of endocytic agents.

[0289] One aspect of the present technology provides a method for allowing or increasing the binding affinity and / or binding valence of an endocytosis agent to a membrane component that mediates endocytosis, dimerization or clustering of a membrane component that mediates endocytosis, and / or conformational changes in a membrane component that mediates endocytosis to enhance the endocytic efficacy and / or efficiency of the agent. In some embodiments, the method refers to forming a multivalent endocytosis agent or using a medicinal chemistry strategy in the art, such as introducing a reversible or irreversible covalent bond or moiety at any appropriate site of the endocytosis agent in any equivalent amount. In certain embodiments, the strategy refers to conjugating a chemical arm containing a reversible or irreversible covalent bond or moiety to the agent via a cleavable or non-cleavable chemical bond or linker, or replacing any bond or moiety on the endocytosis agent with a reversible covalent bond or moiety. The resulting endocytosis agent can form a reversible or irreversible covalent interaction with a membrane component that mediates endocytosis, allow or increase dimerization or clustering of a membrane component that mediates endocytosis, and / or allow or increase conformational changes in a membrane component that mediates endocytosis to enhance the endocytic efficacy and / or efficiency.

[0290] One aspect of the present technology provides a method for increasing the solubility of an endocytosis agent and its binding affinity and / or binding valence to membrane components that mediate endocytosis to enhance endocytosis efficacy and / or efficiency. In some embodiments, the method refers to the use of salt-forming technology in the art, the use of medicinal chemistry strategies in the art to structurally modify the agent and / or form a multivalent endocytosis agent via conjugation with a chargeable or charged chemical arm to allow the solubility of the endocytosis agent and its binding affinity and / or binding valence to membrane components that mediate endocytosis to enhance endocytosis efficacy and / or efficiency. The resulting endocytosis agent can exist in a cationic or anionic form in an aqueous solution at a specific pH value and has increased solubility. The method allows the endocytosis agent to spontaneously have enhanced solubility and permeability via endocytosis for use in disease diagnosis and treatment. In some embodiments, the method refers to the formation of exocytic vesicles in vitro or in vivo to allow for increased stability and solubility of the endocytosis agent.

[0291] One aspect of the present technology provides a method for allowing endocytic uptake of pharmaceutical agents without restrictions on the molecular weight, polarity and lipophilicity of the endocytic agent. In certain embodiments, the present technology provides a method for promoting the endocytic uptake of hydrophilic agents, including but not limited to inorganic compounds, chelates, metal-based compounds and / or polar organic compounds, such as peptide-based compounds. For example, by binding to membrane components that mediate endocytosis via salt bridges and / or hydrogen bond formation, highly polar compounds can be taken up by cells via endocytosis. Another aspect of the present technology provides a method for increasing the binding affinity and / or binding valence to membrane components that mediate endocytosis, and / or dimerization or clustering of membrane components that mediate endocytosis, and / or conformational changes of membrane components that mediate endocytosis to enhance the endocytic efficacy and / or efficiency of polar or hydrophilic agents by preparing charged molecules or salt-forming technologies in the art, forming multivalent endocytic agents via connecting chemical arms, or structural modification using medicinal chemistry strategies in the art (including the introduction of reversible or irreversible covalent bonds or moieties). In certain embodiments, by introducing a moiety that targets the hydrophobic core of CD36 onto, for example, a polar phosphatase inhibitor, the resulting endocytic phosphatase inhibitor agent can have increased binding affinity and / or binding valency to membrane components that mediate endocytosis, induce dimerization and / or clustering of membrane components that mediate endocytosis, and / or induce conformational changes in membrane components that mediate endocytosis, thereby enhancing endocytosis efficacy and / or efficiency. In certain embodiments, by introducing a CD36-binding moiety onto an insulin molecule using a cleavable or non-cleavable bond or moiety, the resulting endocytic insulin agent can be used via oral administration due to increased binding affinity and / or binding valency to membrane components that mediate endocytosis, induce dimerization or clustering of membrane components that mediate endocytosis, and / or induce conformational changes in membrane components that mediate endocytosis, thereby enhancing endocytosis efficacy and / or efficiency. Encapsulation of insulin in exosomes after endocytosis and exocytosis enhances stability. Another benefit of the present technology provides methods that allow endocytic agents to cross cell membranes and / or body barriers via endocytosis, alone or in combination with other mechanisms, including but not limited to passive diffusion, facilitated diffusion, transporter-mediated influx and / or efflux, and paracellular transport.

[0292] One aspect of the present technology provides methods for affecting the metabolic stability of endocytic agents. To meet the stability requirements of pharmaceutical agents used for treatment and diagnosis of subjects, the methods disclosed herein include, but are not limited to, addition or removal of substituents, fragment replacement, cyclization, backbone transitions, bioisostericity, linkerology (study of linker-activity relationships), prodrugs, reduction of overall LogP values, or formation of exocytic vesicles in vitro or in vivo. In certain embodiments, the methods involve deuterium replacement of any hydrogen atom on the endocytic agent. In certain embodiments, the methods involve introduction of surrogates to reduce metabolism at soft spots in the pharmaceutical agent.

[0293] One aspect of the present technology provides methods for preparing and / or using (polypharmacological) endocytic agents for any purpose, wherein at least one target of the (polypharmacological) endocytic agent is a membrane component that mediates endocytosis. Since the binding properties of the endocytic agent to the membrane component that mediates endocytosis can be independent of its binding ability to the intrinsic pharmacological target, it allows the endocytic agent to become a (polypharmacological) compound that spontaneously has binding affinity for both the membrane component that mediates endocytosis for endocytosis and the pharmacological target for intrinsic pharmacological activity without the need for balance. In some embodiments, a method for identifying or generating a (polypharmacological) endocytic agent is to directly structurally modify the agent so that it is easy to bind to the membrane component that mediates endocytosis for endocytosis and the pharmacological target for intrinsic pharmacological activity at the same time. Among them, the process of identifying or generating new (polypharmacological) endocytic and functional targeting agents, classical medicinal chemistry in this field is to spontaneously monitor the activity of both endocytic and biological target modulation efficiency. Structural modifications include, but are not limited to, structural modification of the agent using medicinal chemistry strategies known in the art, formation of multivalent endocytic agents via conjugation with chargeable or charged chemical arms, and formation of exocytic vesicles.

[0294] One aspect of the present technology provides methods for preparing and / or using (polypharmacological) endocytic and exocytic vesicles for transporting (polypharmacological) endocytic and exocytic vesicles across barriers to foreign substances in the human body, such as the blood-retinal barrier, the lung endothelial and epithelial barriers, the skin barrier, and the brain-blood barrier, for any purpose. In some embodiments, a method of preparing and / or using (polypharmacology) endocytic agents and exocytic vesicles is that the endocytic agents and exocytic vesicles bind to receptors or proteins expressed intracellularly in the barrier (such as scavenger receptors, Mfsd2a, GLUT1, fucarin-1, fucarin-2, glutathione transporters, amino acid transporters, transferrin receptors, lactoferrin receptors, low-density lipoprotein receptors, nicotinic acetylcholine receptors, insulin receptors, insulin-like growth factor receptors, integrins and / or CD13 / APN receptors) and are transported across the barrier via the endocytic / exocytic pathway in the form of free endocytic agents, exocytic vesicles, or a mixture of free endocytic agents and exocytic vesicles in any ratio. In some embodiments, a method of preparing and / or using (polypharmacology) endocytic agents and exocytic vesicles is that the endocytic agent is taken up into the cell via endocytosis and encapsulated in intracellular organelles or vesicles (such as endosomes and multivesicular bodies), thereby reducing or preventing efflux transporters (such as P-gp, BCRP and multidrug resistance-associated proteins MRP1, MRP3, MRP4 and MRP6) from pumping the endocytic agent out of the cell. In some embodiments, a method of using the expression of membrane components that mediate endocytosis in a membrane barrier and / or any structural modification of the agent disclosed herein to enhance the efficacy and / or efficiency of endocytic agent endocytosis and the transport of endocytic agents across membrane barriers for any purpose. In particular, a method of using (polypharmacology) endocytic agents and exocytic vesicles is to administer (polypharmacology) endocytic agents and exocytic vesicles by any route for the treatment of ocular, respiratory, skin and CNS conditions and diseases, wherein the (polypharmacology) endocytic agents and exocytic vesicles can be used alone or in combination in any ratio.

[0295] One aspect of the present technology provides a method for transporting endocytic agents across more than one cell layer in the body for any purpose, wherein the endocytic agent can be taken up by cells via endocytosis and can be released in the form of free endocytic agent molecules and / or endocytic agent-vesicle complexes (i.e., exocytic vesicles), wherein the complex comprises the endocytic agent and a lipid bilayer vesicle, including an extracellular vesicle, and the free endocytic agent molecules and / or exocytic vesicles can be taken up again by any (receptor) cell via endocytosis or membrane fusion to exert their effects for any application purpose.

[0296] One aspect of the present technology provides methods for preparing exocytic vesicles in vitro and / or in vivo for any purpose. In particular, one aspect of the present technology provides a "one-step" method for generating and using exocytic vesicles in an animal, wherein, without further processing including agent or vesicle isolation, cells in the body take up an endocytic agent via endocytosis and subsequently secrete exocytic vesicles, and the resulting endogenous exocytic vesicles can be directly used by the body for any purpose. In certain embodiments, "body" or "animal body" refers to the human body. In another embodiment, a method for generating exocytic vesicles is to bind free endocytic agent molecules to extracellular vesicles and / or exocytic vesicles via in situ covalent or non-covalent bonds. In another embodiment, a method for generating exocytic vesicles is to load an endocytic agent into exocytic vesicles in vitro by any technique known in the art. In another embodiment, a method for isolating exocytic vesicles from cells, body fluids, tissues, organs, products, or culture media by any extracellular vesicle isolation technique known in the art.

[0297] One aspect of the present technology provides methods for adjusting the absorption, distribution, metabolism, and excretion (ADME) properties of endocytic agents in animals. In certain embodiments, in order to extend the residence time and / or half-life of a given endocytic agent in an animal, one method is to enhance the endocytic efficacy and / or efficiency of the endocytic agent via the above-mentioned methods to increase the loading of the endocytic agent into exocytic vesicles or the release of the exocytic vesicles via endocytosis, which can reduce or prevent the rapid metabolism and / or excretion of the endocytic agent.

[0298] One aspect of the present technology provides a method for evaluating and / or determining the ADME properties of endocytic agents during drug discovery and development, which involves separating and / or lysing exocytic vesicles by any technology and skill in the art. In certain embodiments, the techniques, skills, and agents that can be used for exocytic vesicle separation processes include, but are not limited to, any suitable method for separating extracellular vesicles (EVs) or cells as listed in publications Brennan K. et al., Scientific Reports, 10, 1039, 2020 and Thanaporn Liangsupree et al., 1636, 461773, 2021. In certain embodiments, the techniques, skills, and agents that can be used for exocytic vesicle lysis processes include, but are not limited to, any suitable method or agent for lysing extracellular vesicles (EVs) or cells or EVs or cell membrane rupture, including freeze-thaw cycles, and any method or agent as listed in publications Prabal Subedi et al., Analytical Biochemistry, 584, 113390, 2019.

[0299] In some embodiments, the method refers to reducing the toxicity of an agent / drug by preparing and using exocytic vesicles from an endosome in vitro or in vivo. Since the endosome can be taken up and released by cells in the form of exocytic vesicles, such as endosome-exosome complexes, wherein the endosome can be present in exosomes, direct contact between free endosome molecules and cells in the body (such as blood cells) is prevented or reduced. In certain embodiments, a method refers to oral administration of an endosome, where cells in the human body (such as cells in the gastrointestinal tract, liver, or tumor) take up the endosome via endocytosis and secrete exocytic vesicles, and the resulting exocytic vesicles have reduced toxicity compared to free endosome molecules. In certain embodiments, a method of reducing toxicity is to adjust the endocytic efficacy and / or efficiency of the agent, load the endosome into exocytic vesicles, and / or release exocytic vesicles by any of the above methods. For example, by attaching or fusing additional chemical arms having binding affinity for membrane components mediating endocytosis to an endocytic agent via a cleavable or non-cleavable chemical bond or linker unit to make it a multivalent compound, the resulting multivalent endocytic agent can have a better affinity for membrane components mediating endocytosis to enhance endocytic efficacy and / or efficiency, enhance the loading of endocytic agents into exocytic vesicles and / or increase the release of exocytic vesicles, and ultimately reduce toxicity because the formation of exocytic vesicles prevents or reduces direct contact between free endocytic agent molecules and cells such as blood cells.

[0300] One aspect of the present technology provides methods for designing or structurally modifying endocytic agents for any purpose using medicinal chemistry strategies in the art, or for targeting specific conformations of membrane components that mediate endocytosis using endocytic agents. In certain embodiments, the present technology provides methods for utilizing the conformational diversity of CD36 on normal cells and cancer cells to design or modify the structure of endocytic agents using medicinal chemistry strategies in the art, or for selecting endocytic agents to target specific conformations of CD36 on cancer cells to enhance anti-tumor efficacy and reduce toxicity. Another benefit of the present technology provides methods for using any method in the art (such as applying mechanical, electrical, thermal, cold, light or radiation stimulation and / or the presence of an endocytic agent) to change the conformation of membrane components that mediate endocytosis, resulting in changes in biological events in the cell and / or changes in the sensitivity of membrane components that mediate endocytosis to endocytic agents.

[0301] One aspect of the present technology provides a general method for improving clinical outcomes. In some embodiments, the method refers to the use of differences in the expression of membrane components that mediate endocytosis for patient stratification, route of administration, and dose selection. At the same time, feedback on clinical endocytosis treatment can be used to adjust the route of administration of the endocytosis agent and / or improve the results of endocytosis treatment. In certain embodiments, the method refers to treating a subject in need of an endocytosis agent. In certain embodiments, the method refers to treating a subject in need of an endocytosis agent by utilizing different expression of membrane components that mediate endocytosis in cells and tissues via special delivery systems, such as local, inhalation, intraperitoneal, intravenous, and oral delivery. For example, CD36-mediated endocytosis is the main pathway for cells to take up nutrients and macromolecules (Jürgen Pohl et al., Molecular Biology of the Cell, 16(1), 24-31, 2005; Nanxia Zhao et al., Advanced NanoBiomed Research, 2(6), 2100120, 2022; Vincenza Cifarelli et al., Comprehensive Physiology, 8(2), 493-507, 2018; Youchun Zeng et al., Journal of Biological Chemistry, 278(46), 45931-45936, 2003). CD36 is highly expressed in intestinal, cancerous cells, and metabolic-related cells, and is upregulated in the central nervous system of patients with pathological changes (Vincenza Cifarelli et al., Comprehensive Physiology, 8(2), 493-507, 2018; Shunjie Bai et al., Translational Psychiatry, 11(16), 2021; Octavian Ioghen et al., European Journal of Neuroscience, 53, 2500-2510, 2021). In addition, receptor-mediated endocytosis and exocytosis pathways are the main pathways for transporting nutrients (such as folic acid and the like) into the brain parenchyma in the form of free nutrients and extracellular vesicles loaded with nutrients (Marcel Grapp et al., Nature Communications, 4, 2123, 2013; Andong Qiu et al., Cell, 127, 917-928, 2006). Thus, the present technology provides, inter alia, a general method to increase the efficacy and reduce the toxicity of more extensively endocytosed agents into the central nervous system following oral or intravenous delivery for therapeutic and prophylactic administration.

[0302] One aspect of the present technology provides a method for identifying a bioactive compound known in the literature that can be taken up by cells via endocytosis. The method comprises adjusting the expression of a membrane component that mediates endocytosis in a cell, tissue, and / or body by editing a membrane component that mediates endocytosis, and then comparing the activity of the bioactive compound in cells, tissues, and / or bodies with and without editing the membrane component that mediates endocytosis. The compound that is taken up via endocytosis has significantly increased or decreased biological activity in cells, tissues, and / or bodies that have endocytosis-mediated membrane component editing compared to the activity of the compound in cells, tissues, and / or bodies that have not endocytosis-mediated membrane component editing.

[0303] One aspect of the present technology provides methods for identifying biological targets in cells and the body using the endocytic agents of the present invention as probes by any technique and skill in the art. For example, the endocytic agents of the present invention having biotin, fluorescence, Halo-tag ligands, SNAP-tag ligands, CLIP-tag ligands, or chemical bonds or moieties that can form covalent bonds with any biological target are particularly suitable for use with microscopic imaging, immunophenotyping, immunoprecipitation, flow cytometry, fluorescence activated cell sorting (FACS), fluorescence resonance energy transfer (FRET) and / or omics (including genomics, epigenomics, transcriptomics, proteomics and metabolomics) analysis to identify biological targets of endocytic agents and derivatives. In particular, one aspect of the present technology provides methods for identifying membrane targets that mediate endocytosis. In a certain embodiment, the method for identifying membrane targets that mediate endocytosis refers to the use of a labeled endocytic agent, followed by administration of an agent and any detection and identification technique in the art. For example, a labeled probe (such as biotin) is attached to any site of the endocytic agent to produce a biotin-labeled endocytic agent. Subsequently, the endocytosis of membrane components that target mediated endocytosis of the endocytic agent can be identified by culturing the cells with biotin-labeled endocytic agents, isolating membrane proteins, and then performing immunoprecipitation, FACS, omics analysis, and protein blot confirmation. In another embodiment, the method for identifying membrane targets that mediate endocytosis refers to using any genome scanning technology in the art to identify membrane targets that mediate endocytosis. For example, comparison of gene expression between cells, tissues, or bodies with different sensitivities to certain endocytic agents can be used to identify membrane targets that mediate endocytosis. In another embodiment, the method for identifying membrane targets that mediate endocytosis refers to using any gene editing technology in the art to identify membrane targets that mediate endocytosis, wherein gene-edited cells, tissues, or bodies may be more sensitive or resistant to endocytic agent treatment. For example, cells that are subjected to gene expression inhibition and activation by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa), respectively, act differently or complementary to certain endocytic agent treatments, which can be used to identify membrane targets that mediate endocytosis.

[0304] One aspect of the present technology provides methods for using and selecting the endocytic agents of the present invention for any purpose based on the various microenvironmental factors of cells and tissues, including but not limited to pH, salinity, oxygen gradients, carbon dioxide gradients, H2O2 gradients, nutrient gradients, and therapeutic compound gradients. For example, due to the acidic environment of cancer cells, endocytic agents with basic groups can be used for tumor-targeted delivery to enhance therapeutic efficacy and reduce toxicity.

[0305] One aspect of the present technology provides methods for using and selecting endocytic agents of the present invention based on the expression of membrane components that mediate endocytosis with different conformations, isoforms (or variants) and / or post-translational modifications such as glycosylation status for any purpose. Another aspect of the present technology provides methods for using and selecting endocytic agents of the present invention based on the expression of cofactors that form complexes with membrane components that mediate endocytosis for any purpose. For example, by targeting specific conformations, isoforms or glycosylation of membrane components or cofactors that mediate endocytosis in cancer cells, endocytic agents can be used to enhance therapeutic function and reduce toxicity in cancer treatment.

[0306] One aspect of the present technology provides a method for activating the endocytic process or increasing the expression of endocytic-mediated membrane components to enhance the endocytic absorption of endocytic agents by regulating input signals (such as the binding of endogenous or exogenous substances to membrane proteins). In certain embodiments, the method refers to the use of endogenous substances (e.g., insulin and derivatives) to bind to membrane proteins (e.g., insulin receptors) to activate the endocytic cycle of membrane components that mediate endocytosis (such as GLUT4) and / or increase the expression of membrane components that mediate endocytosis, thereby resulting in enhanced absorption of endocytic agents via endocytosis. In certain embodiments, the method refers to the use of endocytic agents to simultaneously bind to membrane components that mediate endocytosis and membrane components that do not mediate endocytosis to activate the endocytic process or increase the expression of membrane components that mediate endocytosis, and enhance absorption via endocytosis.

[0307] One aspect of the present technology provides a method for delivering any disease-related membrane component or extracellular material that mediates endocytosis into a cell via endocytosis and destroying or degrading the disease-related membrane component or extracellular material that mediates endocytosis via the endosomal / lysosomal system. In certain embodiments, the disease-related membrane component or extracellular material that mediates endocytosis includes but is not limited to membrane proteins or extracellular proteins, carbohydrates, lipids, pathogens, particles, viruses, fungi, protozoa, bacteria, vectors, cell debris, and another cell.

[0308] One aspect of the present technology provides methods for treating a subject in need of any of the endocytosis agents described herein. Suitably, the method comprises administering to the subject an effective amount of the agent. As used herein, the terms "treating" or "to treat" each mean to alleviate symptoms, temporarily or permanently eliminate the cause of the symptoms, and / or prevent or delay the onset of symptoms of the disease or condition or reverse its progression or severity. Thus, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0309] One aspect of the present technology provides a method for diagnosing a subject in need of any of the endocytosis agents described herein. Suitably, the method comprises administering to the subject an effective amount of the agent. As used herein, the terms "diagnosing" or "to diagnose" each mean to visualize or image a site of pathology, alleviate symptoms, temporarily or permanently eliminate the cause of the symptoms, and / or prevent or delay the appearance of symptoms or reverse the progression or severity of the disease or condition. Thus, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0310] "Subject" is used interchangeably with "patient" or "individual" and refers to an animal in need of treatment, which can be a human or non-human animal. A "subject in need of treatment" includes a subject having a disease, disorder, or condition that is responsive to therapy with an endocytosis agent disclosed herein (alone or in combination with another agent). Preferably, the subject in need of treatment includes, but is not limited to, aging and age-related diseases and conditions, weight management, cancer, central nervous system (CNS) diseases and conditions, cardiovascular disease (CVD), diabetes, eye diseases and conditions, hypertension, immune system diseases, infections, inflammation and inflammation-related diseases and conditions, obesity and obesity-related diseases and conditions, respiratory diseases and conditions, or skin diseases and conditions.

[0311] In certain embodiments, subjects in need of treatment include subjects in need of prevention, treatment and research of aging and age-related diseases and conditions. "Aging" is a physiological process mediated by biological and genetic pathways that is directly associated with lifespan and is the driving force of age-related diseases. Aging mechanisms have been identified, including but not limited to genomic instability, telomere shortening and cellular senescence. For example, aging-related diseases include but are not limited to cardiovascular disease, cancer, diabetes, immune system disorders, hearing loss, macular degeneration and musculoskeletal disorders, such as osteoarthritis.

[0312] In certain embodiments, subjects in need of treatment include those in need of prevention, treatment, and research of cancer. "Cancer" refers to a pathological process that leads to the formation and growth of cancerous or malignant neoplasms, i.e., abnormal tissues that often grow faster than normal tissues through cell proliferation and continue to grow after the stimulus that initiated the new growth has ceased. For example, cancers include, but are not limited to, mesothelioma, leukemias, and lymphomas, such as cutaneous T-cell lymphoma (CTCL), non-cutaneous peripheral T-cell lymphoma, lymphomas associated with human T-lymphotropic virus (HTLV), such as adult T-cell leukemia / lymphoma (ATLL), B-cell lymphoma, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, lymphomas and multiple myeloma, non-Hodgkin lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma, Burkitt lymphoma, adult T-cell leukemia lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), or hepatocellular carcinoma. Further examples include myelodysplastic syndrome; solid tumors in children; such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft tissue sarcomas; common solid tumors in adults, such as head and neck cancer (e.g., oral cancer, laryngeal cancer, nasopharyngeal cancer, and esophageal cancer), genitourinary cancer (e.g., prostate cancer, bladder cancer, kidney cancer, uterine cancer, ovarian cancer, testicular cancer), lung cancer (e.g., small cell cancer and non-small cell cancer), breast cancer (e.g., triple-negative breast cancer (TNBC)), pancreatic cancer, melanoma and other skin cancers, stomach cancer, brain tumors, tumors associated with Gorlin's syndrome (e.g., medulloblastoma or meningioma), or liver cancer.

[0313] Additional exemplary forms of cancer that can be prevented or treated by endocytosed agents include, but are not limited to, skeletal or smooth muscle cancer, stomach cancer, small intestine cancer, rectal cancer, salivary gland cancer, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer, pituitary cancer, colon cancer, familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer, lip cancer, laryngeal cancer, carcinoma, tongue cancer, salivary gland cancer, stomach cancer, thyroid cancer (medullary and papillary thyroid cancer), kidney cancer, carcinoma, renal parenchymal cancer, kidney cancer, cervical cancer, uterine corpus cancer, endometrial cancer, choriocarcinoma, testicular cancer, urinary tract cancer, brain tumors (such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumor), gallbladder cancer, bronchial cancer, basal cell tumor, teratoma, retinoblastoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing's sarcoma sarcoma), plasmacytoma, melanoma, nasopharyngeal carcinoma (NPC), microsatellite stable colorectal cancer (mssCRC), thymoma, carcinoid, acute myeloid leukemia and gastrointestinal stromal tumor (GIST), breast cancer, triple-negative breast cancer (TNBC), small cell lung cancer, non-small cell lung cancer, prostate cancer, castration-resistant prostate cancer (CRPC) or metastatic castration-resistant prostate cancer (mCRPC).

[0314] In certain embodiments, subjects in need of treatment include subjects in need of prevention, treatment, and research of cardiovascular disease. "Cardiovascular disease" refers to a group of conditions affecting the heart and blood vessels. For example, cardiovascular disease includes, but is not limited to, coronary heart disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis, or pulmonary embolism.

[0315] In certain embodiments, the subject in need of treatment includes the subject in need of prevention, treatment and research of central nervous system (CNS) diseases and the patient's condition." CNS diseases and the patient's condition " refers to a group of extensive nervous system disorders affecting the structure or function of the brain or spinal cord (together constituting the central nervous system). For example, CNS diseases include but are not limited to brain tumors, neurodegenerative diseases, such as Alzheimer's disease (Alzheimer disease), frontotemporal dementia, Pick's disease (Pick disease), progressive supranuclear palsy, corticobasal degeneration, vascular dementia, Parkinson's disease (Parkinson disease), dementia with Lewy bodies (dementia with Lewy bodies), Huntington's disease (Huntington disease), spinocerebellar ataxia, Friedrich ataxia (Friedrich ataxia), ataxia telangiectasia, amyotrophic lateral sclerosis (ALS), bulbar muscular atrophy or spinal muscular atrophy.

[0316] In certain embodiments, subjects in need of treatment include subjects in need of prevention, treatment, and research of diabetes. "Diabetes" refers to a group of diseases in which the body's ability to produce or respond to insulin is impaired, leading to abnormal carbohydrate metabolism and elevated glucose levels in the blood and urine. For example, diabetes includes, but is not limited to, type 2 diabetes, gestational diabetes, prediabetes, monogenic diabetes, cystic fibrosis-related diabetes, or drug- or chemical-induced diabetes.

[0317] In certain embodiments, subjects in need of treatment include subjects in need of prevention, treatment, and research of ocular diseases and conditions. "Ocular diseases and conditions" refers to any disease or condition that affects the human eye. For example, ocular diseases include, but are not limited to, age-related macular degeneration (AMD), amblyopia, anophthalmia and microphthalmia, astigmatism, Behcet's disease ( disease), Bietti's crystalline dystrophy, blepharitis, blepharospasm, cataracts, central retinal vein occlusion (CRVO), cerebral visual impairment (CVI), coloboma, color blindness, convergence insufficiency, corneal conditions, diabetic retinopathy, dry eye, eye cancer, farsightedness (hyperopia), floaters, glaucoma, Graves' eye disease, idiopathic intracranial hypertension, low vision, macular edema, macular hole, macular pucker, nearsightedness (myopia), ocular histoplasmosis syndrome (OHS), red eye, presbyopia, rare diseases, refractive error, retinal detachment, retinitis pigmentosa, retinoblastoma, retinopathy of prematurity, Stargardt disease, Usher syndrome, uveitis, or vitreous detachment.

[0318] In certain embodiments, subjects in need of treatment include those in need of prevention, treatment, and research of hypertension. "Hypertension" refers to a medical condition in which blood pressure in the arteries is persistently elevated. For most adults, hypertension is present if resting blood pressure is persistently at or above 120 / 80 mmHg, 130 / 80 mmHg, or 140 / 90 mmHg. For example, hypertension includes, but is not limited to, essential hypertension or secondary hypertension.

[0319] In certain embodiments, subjects in need of treatment include subjects in need of prevention, treatment, and research of immune system diseases and conditions. "Immune system diseases and conditions" refer to immunodeficiency and autoimmune disorders. Among them, "autoimmune disease" refers to a condition caused by an abnormal immune response to a functional body part. A functional body part refers to any body part of an animal. For example, autoimmune diseases include, but are not limited to, rheumatoid arthritis (RA), psoriasis / psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), inflammatory bowel disease, Addison's disease, Graves' disease, Sjögren's syndrome ( syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, or celiac disease.

[0320] In certain embodiments, subjects in need of treatment include subjects in need of prevention, treatment and research of inflammation and inflammation-related diseases and conditions. "Inflammation" is when the immune system attacks the body's own tissues, causing inflammation. Many conditions may be associated with inflammation (especially chronic inflammation). For example, inflammation-related diseases include but are not limited to Alzheimer's disease, asthma, cancer, CVD, rheumatoid arthritis (RA), ankylosing spondylitis (AS) or stroke.

[0321] In certain embodiments, subjects in need of treatment include those in need of prevention, treatment, and research of weight management, obesity, and obesity-related diseases and conditions. "Obesity" refers to a physical condition in which a body mass index (BMI) is 30.0 or higher. Obesity-related diseases include, but are not limited to, type 2 diabetes, CVD, fatty liver disease, or cancer. Weight management refers to techniques and physiological processes that help individuals achieve and maintain a specific weight.

[0322] In certain embodiments, the endocytic agents described herein can be used to prevent, treat, and study respiratory diseases and conditions. "Respiratory diseases" refers to diseases that affect the lungs and other parts of the respiratory system. For example, respiratory diseases include, but are not limited to, asthma, chronic obstructive pulmonary disease (COPD), lung disease, pneumonia, idiopathic pulmonary fibrosis, or lung cancer.

[0323] In certain embodiments, subjects in need of treatment include subjects in need of prevention, treatment, and research of skin diseases and conditions. "Skin diseases and conditions" refers to any condition that affects the skin. For example, skin diseases and conditions include, but are not limited to, acanthosis nigricans, acne, keloid acne nuchae, acne scars, actinic keratosis, alopecia areata, tinea pedis, atopic dermatitis, basal cell carcinoma, bed bugs, birthmarks, boils and styes, botulinum toxin, bullous pemphigoid, cellulitis, central centrifugal cicatricial alopecia (CCCA), chemical peels, chicken pox, cold sores, and eczema. sore), contact dermatitis, cradle cap, cutaneous T-cell lymphoma, dandruff, dermatofibrosarcoma protuberans (DFSP), diabetes-related skin conditions, diaper rash, dry skin, dyshidrotic eczema, epidermolysis bullosa, female pattern hair loss, folliculitis, frontal fibrosing alopecia, genital herpes, genital warts, granuloma annulare, hair loss, hand, foot and mouth disease, head lice, heart-related skin conditions, herpes simplex, hidradenitis suppurativa, urticaria, hyperhidrosis, ichthyosis vulgaris, imiquimod, impetigo, isotretinoin, keloids, keratosis pilaris, kidney-related skin conditions, laser-related skin conditions, leprosy, lichen planus, lupus, Lyme disease, melanoma, melasma, Merkel cell carcinoma carcinoma), moles, molluscum contagiosum, monkeypox rash, onychomycosis, neurodermatitis, nickel allergy, nummular dermatitis, ocular rosacea, pemphigus, perioral dermatitis, pityriasis rosea, poison ivy, oak, and sumac-related skin conditions, prurigo nodularis, psoriasis, psoriatic arthritis, rash, ringworm, rosacea, sarcoidosis, scabies, scalp psoriasis, scars, scleroderma, sebaceous gland carcinoma, seborrheic dermatitis, seborrheic keratosis, shingles, skin biopsy, skin cancer, squamous cell carcinoma, stasis dermatitis, stretch marks, syphilis, skin conditions related to thyroid disease, tinea versicolor, vitiligo, warts, wounds, xeroderma, or pigmentosum.

[0324] In some embodiments, subjects in need of treatment include subjects in need of sensitization to a bioactive agent (such as an anti-aging agent, an anti-aging-related disease agent, an anti-cancer agent, an anti-cardiovascular disease agent, an anti-diabetic agent, an anti-ocular disease agent, an anti-hypertensive agent, an anti-immune system disease agent, an anti-infective agent, an anti-inflammatory agent, an anti-inflammatory-related disease agent, an anti-CNS disease agent, a weight management agent, an anti-obesity agent, an anti-obesity-related disease agent, an anti-respiratory disease agent, an anti-skin disease and condition agent). The terms "sensitize" and "sensitizing" refer to making a subject or cell more susceptible or responsive to the biological effects of a second agent (e.g., a bioactive agent) (e.g., promoting or delaying a certain aspect of cell function, including but not limited to cell secretion, cell division, cell growth, proliferation, invasion, angiogenesis, necrosis or apoptosis) by administering a first agent. Sensitization of target cells by a first agent can be measured as the difference in the expected biological effect (e.g., promotion or delay of some aspect of cellular function, including but not limited to cell growth, proliferation, invasion, angiogenesis, or apoptosis) observed following administration of a second agent with and without administration of the first agent.

[0325] In some embodiments, the endocytosis agents described herein may be administered with one or more bioactive agents, including anti-aging agents, anti-aging-related disease agents, anti-cancer agents, anti-cardiovascular disease agents, anti-diabetic agents, anti-ocular disease agents, anti-hypertensive agents, anti-immune system disease agents, anti-infective agents, anti-inflammatory agents, anti-inflammatory-related disease agents, anti-CNS disease agents, anti-obesity agents, anti-obesity-related disease agents, anti-respiratory disease agents, and anti-skin disease and condition agents. The term "bioactive agent" is used to describe an agent that has biological activity to help achieve the intended therapy, inhibition, and / or prevention (prevention / prophylaxis) for which the compounds of the present invention are used. Exemplary bioactive agents include anti-cancer agents. "Anti-cancer agent" means a compound or composition that can be combined with an endocytosis agent to treat cancer, inhibit cancer cell growth or proliferation, or kill cancer cells. Suitably, the endocytosis agent can be administered before, during, or after administration of the bioactive agent. The endocytosis agent can sensitize the subject to the bioactive agent. This allows improving the therapeutic efficacy of the bioactive agent, reducing the effective amount of the bioactive agent required to achieve a desired effect or reducing the duration of treatment with the bioactive agent.

[0326] While the present disclosure demonstrates the utility of this technology with various probes or therapeutic endocytic agents, the technology is not limited to those probes or therapeutic endocytic agents. In some embodiments, endocytic agents include, but are not limited to, immunoconjugates, drugs, prodrugs, cytotoxic agents, pro-apoptotic agents, toxins, nucleases (including DNA enzymes and RNA enzymes), hormones, vitamins, immunomodulators, chelating agents, boron compounds, photoactive agents, radionuclides, oligonucleotides, interfering DNA, RNA, siRNA, RNAi, anti-angiogenic agents, protein inhibitors, protein activators, molecular glues, degraders, chemotherapeutic agents, cytokines, chemokines, amino acids, peptides, deuterated derivatives thereof, or combinations thereof.

[0327] As used herein, the term "effective amount" refers to the amount or dose of a compound that provides the desired effect, such as after single or multiple dose administration to a subject. With respect to sensitization, an effective amount will refer to the amount of a therapeutic agent that results in sensitization of a subject or cell as described above.

[0328] As used herein with respect to weight management, the term "specific weight" refers to the weight at which an individual maintains or loses their desired weight.

[0329] An effective amount can be determined by the attending diagnostician, who is skilled in the art, using known techniques and by observing results obtained under similar circumstances.

[0330] In some embodiments, the endocytic agents utilized in the methods disclosed herein can be formulated into pharmaceutical compositions comprising: (a) a therapeutically effective amount of one or more endocytic agents described herein and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. Such compositions take any pharmaceutically acceptable physical form; illustratively, they can be pharmaceutical compositions for oral administration. Such pharmaceutical compositions contain an effective amount of the disclosed endocytic agents that is related to the daily dose of the agent to be administered. Each dosage unit contains the daily dose of a given endocytic agent, or each dosage unit contains a portion of the daily dose, such as half or one-third of the dose. The amount of each endocytic agent to be contained in each dosage unit may depend in part on the characteristics of the endocytic agent selected for treatment and other factors, such as the indication for which it is used. The pharmaceutical compositions disclosed herein can be formulated using well-known procedures to provide rapid, sustained, or delayed release of the endocytic agent following administration to a patient. The endocytic agents used in accordance with the methods disclosed herein can be administered as a single compound or a combination of compounds. For example, an endocytic agent having anti-cancer activity can be administered as a single compound or in combination with another compound that also promotes anti-cancer activity or has a different pharmacological activity.

[0331] In certain embodiments, the endocytic agent utilized in the methods disclosed herein can be loaded via extracellular vesicles (EVs) for any purpose to form EVs loaded with the endocytic agent in vitro, wherein the extracellular vesicles include but are not limited to exosomes, microvesicles, extracellular granules, cancer bodies, prostate bodies, and apoptotic bodies. It is understood that EVs loaded with endocytic agents can be obtained by any in vitro technique for the generation, separation, and modification of drug-loaded EVs in the art, including but not limited to methods such as those shown in publications Shuang Du et al., Journal of Nanobiotechnology, 21, 231, 2023 and publications Inge Katrin Herrmann et al., Nature Nanotechnology, 16, 748-759, 2021. In some embodiments, the endocytic agent utilized in the methods disclosed herein can be loaded into exosomes as nanoparticles by microfluidic droplet-based electroporation (μDES), which can result in enhanced stability, biocompatibility, transportability, and / or targeting capabilities relative to free endocytic agents.

[0332] In certain embodiments, the endocytic agents (including exocytic vesicles) utilized in the methods disclosed herein can be formulated with human serum albumin in vitro or in vivo. Exemplary formulations containing human serum albumin are complexes at a conjugate:albumin molar ratio of 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the endocytic agents used in the methods disclosed herein can be formulated with albumin into nanoparticles, microparticles, albumin-coated liposomes, albumin microbubbles, and albumin nanocapsules. Exemplary nanoparticles range in size from 1 to 100 nm. In further embodiments, the endocytic agent-albumin complex can be used in combination with any of the nanoparticle technologies herein. Exemplary methods for formulation with human serum albumin include, but are not limited to, methods as shown in the publications Ella N. Hoogenboezem et al., Advanced Drug Delivery Reviews, 130, 73-89, 2018; Cassandra E. Callmann et al., Journal of American Chemistry Society, 141(30)11765-11769, 2019; and Shrawani Lamichhane et al., Archives of Pharmacal Research, 43, 118-133, 2020. Formulation of an endocytic agent with albumin in vitro or in vivo can 1) prolong the residence time and / or half-life of a given endocytic agent or exocytic vesicle in an animal compared to the free endocytic agent, 2) increase water solubility, 3) increase the penetration of the endocytic agent across cell membranes and membrane barriers (such as the blood-retinal barrier, lung endothelial and epithelial barriers, skin barrier, and brain-blood barrier), 4) be used to deliver the endocytic agent to specific organ systems, such as the liver and brain, 5) be used to deliver the endocytic agent to specific body tissues, such as tumors, and / or 6) reduce the toxicity of the endocytic agent in an animal.

[0333] In certain embodiments, the endocytic agents (including exocytic vesicles) utilized in the methods disclosed herein can be formulated into pharmaceutical compositions. The term "pharmaceutical composition" refers to an agent of the present disclosure, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, and at least one pharmaceutically acceptable carrier, in a form suitable for oral or parenteral administration.

[0334] In certain embodiments, the endocytic agent utilized in the methods disclosed herein can be formulated as a pharmaceutical composition comprising a carrier. For example, the carrier can be selected from the group consisting of proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

[0335] In certain embodiments, the endocytic agent in the methods disclosed herein can be formulated into a pharmaceutical composition comprising one or more binders, diluents, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents.

[0336] Suitable diluents include pharmaceutically acceptable inert fillers such as microcrystalline cellulose, lactose, dibasic calcium phosphate, sugars, and mixtures of any of the foregoing.

[0337] Suitable disintegrants include lightly cross-linked polyvinyl pyrrolidone, corn starch, potato starch, corn starch and modified starches, cross-linked sodium carboxymethylcellulose, cross-povidone, sodium carboxymethyl starch, and mixtures thereof.

[0338] Examples of effervescent agents are effervescent couples, such as an organic acid and a carbonate or bicarbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0339] In some embodiments, the endocytosis agent utilized in the methods disclosed herein can be used alone or can be formulated into a pharmaceutical composition for administration by any appropriate route, such as oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes, intraperitoneal injection, microneedle patch and topical administration via eye drops. Such formulations can be prepared by any method known in the art of pharmaceutics, for example, by associating the active ingredient with a carrier or excipient. Preferably, the endocytosis agent or composition is administered topically, orally, intraperitoneally, intravenously, via inhalation or via microinjection.

[0340] In certain embodiments, the endocytic agents utilized in the methods disclosed herein can be administered in conventional dosage forms prepared by combining the endocytic agent with a standard pharmaceutical carrier or diluent according to conventional procedures well known in the art. These procedures involve mixing, granulating, and compressing or dissolving the ingredients as appropriate depending on the desired formulation.

[0341] In certain embodiments, the endocytic agent utilized in the methods disclosed herein can be formulated as a pharmaceutical composition in the form of a solid dosage form, although any pharmaceutically acceptable dosage form can be used. Exemplary solid formulations include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid formulation can be, for example, a fast-dissolving dosage form, a controlled-release dosage form, a lyophilized dosage form, a delayed-release dosage form, an extended-release dosage form, a pulsed-release dosage form, a mixed immediate-release and controlled-release dosage form, or a combination thereof.

[0342] In certain embodiments, the endocytic agents utilized in the methods disclosed herein can be practiced using solvates of the compounds or their salts, prodrugs, esters, and / or amides. Solvate forms include ethanol solvates, hydrates, and the like. "Hydrate" means a solvate in which the solvent molecule is water.

[0343] Formulations may be presented in unit-dose or multi-dose containers.

[0344] "Salt" means an ionic form of a parent compound, or the product of the reaction of a parent compound with a suitable acid or base to prepare an acid salt or basic salt of the parent compound. Typically, salts are prepared by reacting the free base or acid parent compound with a stoichiometric amount or an excess of the desired salt-forming inorganic or organic acid or base in a suitable solvent or various combinations of solvents.

[0345] The counterion forming part of any endocytosis agent salt disclosed herein may not be critical to the activity of the compound, so long as the salt is pharmacologically acceptable and so long as the counterion does not impart undesirable properties to the salt as a whole. Undesirable properties include undesirable solubility or toxicity.

[0346] "Prodrug" or "prodrug derivative" means a covalently bonded derivative or carrier of a parent compound or active drug substance that undergoes at least some biotransformation before exhibiting its pharmacological eff...

Claims

1. A compound comprising a cleavable or non-cleavable chemical bond or linker unit With chemical arm Connected agent or probe The compound has binding affinity for a membrane component that mediates endocytosis.

2. The compound according to claim 1, wherein the binding affinity of the compound to the membrane component mediating endocytosis is D Less than 20.0mM.

3. The compound according to any one of claims 1 to 2, wherein the compound is represented by formula (I): , wherein m, n and p represent integers from 0 to 100. The compound according to claim 3 , wherein m, n and p are 1.

5. The compound of any one of claims 1-4, wherein the compound comprises an agent that is a degrader, stabilizer, inhibitor, modulator, or activator.

6. The compound of claim 5, wherein the degrader is a PROTAC.

7. The compound of any one of claims 1-4, wherein the compound comprises an agent that is a protein binding agent.

8. The compound of claim 7, wherein the protein binding agent has a binding affinity K of D Less than 20.0mM.

9. The compound of claims 7-8, wherein the protein is an extracellular protein, an intracellular protein, an integral membrane protein, a peripheral membrane protein, a lipid-anchored protein, a globular protein, or a glycoprotein.

10. The compound of any one of claims 1-4, wherein the compound comprises a probe that is a diagnostic agent.

11. The compound of claim 10, wherein the diagnostic agent comprises a detectable label.

12. The compound of any one of claims 1-11, wherein the chemical arm is an atom, an agent, a probe, or a portion of an agent or a binding agent or a probe.

13. The compound of claim 12, wherein the chemical arm has a binding affinity K of 0.001 to the membrane component mediating endocytosis. D Less than 20.0mM.

14. The compound according to any one of claims 1 to 13, wherein the membrane component mediating endocytosis is a cell membrane lipid, carbohydrate or protein.

15. The compound according to any one of claims 1 to 13, wherein the membrane components mediating endocytosis are glycolipids, glycoproteins, phospholipids, ceramides and cholesterol.

16. The compound according to any one of claims 1 to 13, wherein the membrane component mediating endocytosis is a glycolipid, or a glycoprotein comprising 2 to 100 linear or branched monosaccharide units.

17. The compound of any one of claims 1-13, wherein the membrane component that mediates endocytosis is an integral membrane protein, a peripheral membrane protein, a lipid-anchored protein, a globular protein, or a glycoprotein.

18. A pharmaceutical composition comprising a compound according to any one of claims 1 to 17 and a pharmaceutically acceptable excipient, carrier or diluent.

19. A nanostructure comprising a liquid or cytoplasm enclosed by a lipid bilayer, a membrane component that mediates endocytosis, and a compound according to any one of claims 1 to 17.

20. The nanostructure of claim 19, wherein the binding affinity of the nanostructure to the membrane component mediating endocytosis is D Less than 20.0mM.

21. A method of preparing the nanostructure of claims 19-20, comprising contacting the compound of any one of claims 1-17 with a cell or vesicle comprising a membrane component that mediates endocytosis.

22. The method of claim 21, wherein the compound is contacted in vivo with cells or vesicles comprising membrane components that mediate endocytosis.

23. The method of claim 21, wherein the compound is contacted in vitro or ex vivo with cells or vesicles comprising the membrane component that mediates endocytosis.

24. A method for internalizing a compound within a cell, comprising contacting the compound according to any one of claims 1 to 17 with a cell comprising a membrane component that mediates endocytosis.

25. A method for internalizing a compound within a cell, comprising contacting the nanostructure according to any one of claims 19-20 with a cell comprising a membrane component that mediates endocytosis.

26. A method for isolating a compound from a nanostructure according to any one of claims 19-20, comprising lysing the nanostructure and isolating the compound from the lysate.

27. A method for determining the qualitative or quantitative presence of a compound or nanostructure according to any one of claims 1 to 20 in a cell, body, liquid or medium, comprising centrifuging a sample comprising the cell, body, liquid or medium and detecting the compound or nanostructure.

28. A method for isolating a compound or nanostructure according to any one of claims 1 to 20 from a cell, a body, a liquid or a medium, comprising centrifuging a sample comprising the cell, body, liquid or medium.

29. The method of any one of claims 27-28, wherein the medium is a cell culture medium, a tissue culture medium, an organ culture medium, a body fluid, a tissue or an organ.

30. A method of treating a subject, comprising administering to a subject in need thereof a compound or nanostructure according to any one of claims 1-20.

31. The method of claim 30, wherein the effective amount of the compound or nanostructure for treating the subject is less than the effective amount of the agent or the probe in the absence of the chemical arm for treating the subject.

32. The method of claim 30, wherein the effective amount of the compound or nanostructure for treating the subject is equal to the effective amount of the agent or the probe for treating the subject in the absence of the chemical arm.

33. The method of claim 30, wherein the effective amount of the compound or nanostructure for treating the subject is greater than the effective amount of the agent or the probe in the absence of the chemical arm for treating the subject.

34. A method for identifying an endocytosis agent, comprising contacting a compound with a first cell and a second cell, wherein the presence of a membrane component that mediates endocytosis in the second cell is modulated relative to the first cell, and comparing the activity of the compound in contact with the first cell with the activity of the compound in contact with the second cell to determine that the compound is the endocytosis agent.

35. The method of claim 34, wherein the presence of the endocytosis-mediating membrane component is less in the second cell than in the first cell.

36. The method of claim 35, wherein the presence of the endocytosis-mediating membrane component of the second cell has been reduced by gene editing, knockdown, or silencing.

37. The method of claim 34, wherein the presence of the membrane component that mediates endocytosis is greater in the second cell than in the first cell.

38. The method of claim 37, wherein the presence of the endocytosis-mediating membrane component of the second cell has been increased by gene editing or introduction of DNA or RNA encoding the endocytosis-mediating membrane component.

39. The method of any one of claims 34-38, wherein the compound is contacted with the first cell and the second cell in vivo.

40. The method of any one of claims 34-38, wherein the compound is contacted with the first cell and the second cell in vitro or ex vivo.

41. The method of any one of claims 34-40, further comprising determining the binding affinity, K, of the compound for the membrane component mediating endocytosis. D Less than 20.0mM.

42. The method of any one of claims 34-41, further comprising administering the compound or nanostructure to a subject in need thereof.

43. A method for identifying a membrane component that mediates endocytosis, comprising contacting a cell with a compound or nanostructure according to any one of claims 1 to 29, wherein the compound comprises a detectable label and wherein the membrane component that mediates endocytosis in the cell can be identified by determining the interaction between the membrane component that mediates endocytosis and the detectable label.

44. A method for identifying a membrane component that mediates endocytosis, comprising comparing the sensitivity of a first cell and a second cell to treatment with a compound or nanostructure according to any one of claims 1-20, and comparing the genomic expression or protein abundance of the membrane component in the first cell and the second cell, wherein the increased sensitivity of the first cell or the second cell to the compound or nanostructure identifies the membrane component that mediates endocytosis.

45. The method of claim 44, wherein the first cell having a higher abundance of the membrane component is more sensitive than the second cell having a lower abundance of the membrane component or no membrane component.

46. ​​The method of claim 44, wherein the first cell having a higher expression of the membrane component is more sensitive than the second cell having a lower expression or no expression of the membrane component.

47. The method of any one of claims 44-46, further comprising determining the binding affinity, K, of the membrane component mediating endocytosis to the compound or nanostructure of any one of claims 1-25. D Less than 20.0mM.

48. The method of claims 44-47, wherein the sensitivity of the first cell and the second cell is determined by evaluating a change in a biological process when the first cell and the second cell are contacted with the compound or nanostructure.

49. A method for selecting a subject for treatment with a compound or nanostructure having binding affinity for a membrane component that mediates endocytosis according to any one of claims 1-20, comprising determining the qualitative or quantitative presence of the membrane component that mediates endocytosis in a sample obtained from the intended subject, and administering the compound or nanostructure to the intended subject when the membrane component that mediates endocytosis is present in the sample.

50. The method of claim 49, wherein the route of administration or effective amount of the compound or nanostructure is determined by the qualitative or quantitative presence of membrane components that mediate endocytosis in the sample.

51. The method of any one of claims 49-50, wherein the binding affinity of the compound or nanostructure to the membrane component mediating endocytosis is less than 20 mM.

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